U.S. patent application number 13/382953 was filed with the patent office on 2013-01-24 for optimised coding sequence and promoter.
This patent application is currently assigned to UCL BUSINESS PLC. The applicant listed for this patent is Andrew Davidoff, John Gray, John McVey, Amit Nathwani, Adrian Thrasher, Edward Tuddenham, Natalie Ward. Invention is credited to Andrew Davidoff, John Gray, John McVey, Amit Nathwani, Adrian Thrasher, Edward Tuddenham, Natalie Ward.
Application Number | 20130024960 13/382953 |
Document ID | / |
Family ID | 41022350 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130024960 |
Kind Code |
A1 |
Nathwani; Amit ; et
al. |
January 24, 2013 |
OPTIMISED CODING SEQUENCE AND PROMOTER
Abstract
An optimized coding sequence of human blood clotting factor
eight (VIII) and a promoter may be used in vectors, such as rAAV,
for introduction of factor VIII, and/or other blood clotting
factors and transgenes. Exemplary of these factors and transgenes
are alpha-1-antitrypsin, as well as those involved in the
coagulation cascade, hepatocye biology, lysosomal storage, urea
cycle disorders, and lipid storage diseases. Cells, vectors,
proteins, and glycoproteins produced by cells transformed by the
vectors and sequence, may be used in treatment.
Inventors: |
Nathwani; Amit; (London,
GB) ; Ward; Natalie; (London, GB) ; Thrasher;
Adrian; (London, GB) ; Tuddenham; Edward;
(London, GB) ; McVey; John; (Beaconsfield, GB)
; Gray; John; (Memphis, TN) ; Davidoff;
Andrew; (Memphis, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nathwani; Amit
Ward; Natalie
Thrasher; Adrian
Tuddenham; Edward
McVey; John
Gray; John
Davidoff; Andrew |
London
London
London
London
Beaconsfield
Memphis
Memphis |
TN
TN |
GB
GB
GB
GB
GB
US
US |
|
|
Assignee: |
UCL BUSINESS PLC
London
TN
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
Memphis
THROMBOSIS RESEARCH INSTITUTE
Chelsea
|
Family ID: |
41022350 |
Appl. No.: |
13/382953 |
Filed: |
July 8, 2010 |
PCT Filed: |
July 8, 2010 |
PCT NO: |
PCT/US2010/041378 |
371 Date: |
April 18, 2012 |
Current U.S.
Class: |
800/16 ;
435/320.1; 435/325; 435/358; 435/363; 514/44R; 536/23.5; 536/24.1;
800/14; 800/17; 800/18 |
Current CPC
Class: |
C12N 2710/10041
20130101; A61P 7/04 20180101; C12N 15/86 20130101; C12N 2800/22
20130101; A61K 38/37 20130101; C12N 2710/14143 20130101; A61K
48/0058 20130101; C12N 2830/008 20130101; C07K 14/755 20130101;
A61K 38/00 20130101; C12N 2750/14143 20130101; C12N 7/00
20130101 |
Class at
Publication: |
800/16 ;
435/320.1; 435/325; 435/363; 800/14; 800/18; 800/17; 536/23.5;
536/24.1; 514/44.R; 435/358 |
International
Class: |
C12N 5/10 20060101
C12N005/10; A01K 67/027 20060101 A01K067/027; C12N 15/864 20060101
C12N015/864; C12N 15/113 20100101 C12N015/113; A61K 31/7088
20060101 A61K031/7088; A61P 7/04 20060101 A61P007/04; C12N 15/85
20060101 C12N015/85; C12N 15/12 20060101 C12N015/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2009 |
GB |
0911870.4 |
Claims
1. An isolated nucleic acid molecule comprising a nucleotide
sequence having at least 75% homology to the nucleotide sequence of
SEQ ID NO: 1 and which encodes functional factor VIII.
2. The nucleic acid molecule of claim 1 having at least 85%
homology to the nucleotide sequence of SEQ ID NO: 1.
3. The nucleic acid molecule of claim 1, which encodes a protein
comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 21 having
between 0 and 10 amino acid changes thereto.
4. The nucleic acid molecule of claim 3, which encodes a protein
comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 21.
5. The nucleic acid molecule of claim 1 comprising a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:7.
6. The nucleic acid molecule of claim 1 further comprising a
nucleotide sequence having at least 85% homology to the nucleotide
sequence of SEQ ID NO: 3.
7. A vector comprising a nucleic acid molecule according to claim
1.
8. A host cell comprising the nucleic acid molecule of claim 1 or
the vector of claim 7.
9. (canceled)
10. A transgenic animal comprising cells comprising the nucleic
acid molecule of claim 1.
11. A method of treating haemophilia comprising administering a
vector according to claim 7 to a patient suffering from
haemophilia.
12. (canceled)
13. (canceled)
14. A method for delivery of a nucleotide sequence encoding a
functional factor VIII to a subject, which method comprises
administering to the said subject a nucleic acid molecule according
to claim 1.
15. A promoter comprising a nucleotide sequence having at least 85%
homology to the nucleotide sequence of SEQ ID NO: 3.
16. The promoter of claim 15, wherein the promoter is less than 400
bp.
17. A vector comprising a nucleic acid molecule according to claim
15.
18. A host cell comprising the promoter of claim 15 or the vector
of claim 17.
19. (canceled)
20. A transgenic animal comprising cells comprising the promoter of
claim 15 or the vector of claim 17.
21. A method for the preparation of a parvoviral gene delivery
vector, the method comprising the steps of: (a) providing an insect
cell comprising one or more nucleic acid constructs comprising: (i)
a nucleic acid molecule of claim 1 that is flanked by at least one
parvoviral inverted terminal repeat nucleotide sequence; (ii) a
first expression cassette comprising a nucleotide sequence encoding
one or more parvoviral Rep proteins which is operably linked to a
promoter that is capable of driving expression of the Rep
protein(s) in the insect cell; (iii) a second expression cassette
comprising a nucleotide sequence encoding one or more parvoviral
capsid proteins which is operably linked to a promoter that is
capable of driving expression of the capsid protein(s) in the
insect cell; (b) culturing the insect cell defined in (a) under
conditions conducive to the expression of the Rep and the capsid
proteins.
22. The method of claim 21 further comprising the step of:
recovering the parvoviral gene delivery vector.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an optimised coding sequence of
human blood clotting factor eight (VIII) and a new promoter, which
may be used in such vectors as rAAV for introduction of factor
VIII, other blood clotting factors and transgenes including those
involved in the coagulation cascade, hepatocytes biology, lysosomal
storage, and urea cycle disorders, lipid storage disease,
alpha-1-antitrypsin, into cells to bring about expression thereof.
The invention also relates to cells transformed with such vectors,
proteins and glycoproteins produced by such cells, transgenically
modified animals containing cells modified using the vectors and
methods of treatment utilising the vectors in a gene replacement
approach and using proteins and glycoproteins produced by cells
transformed with the vectors in a more conventional approach.
BACKGROUND OF THE INVENTION
[0002] The inventors are interested in developing a safe and
efficient gene transfer strategy for the treatment of haemophilia A
(HA), the most common inherited bleeding disorder. This would
represent a major clinical advance with significant implications
for other congenital disorders that lack effective treatment. The
inventors have already developed a promising gene therapy approach
for haemophilia B using recombinant adeno-associated viral (rAAV)
vectors. Haemophilia A poses several new challenges due to the
distinct molecular and biochemical properties of human factor VIII
(hFVIII), a molecule that is mutated in this disease. These include
the relatively large size of the hFVIII cDNA and the fact that
hFVIII protein expression is highly inefficient. The inventors have
begun to address some of these limitations through advances in
vector technology and the development of a novel more potent hFVIII
variant (codop-hFVIII) that can be efficiently packaged into
rAAV.
[0003] Haemophilia A (HA) is an X-linked bleeding disorder that
affects approximately 1 in 5,000 males, that is caused by mutations
in the factor VIII (FVIII) gene, which codes for an essential
cofactor in the coagulation cascade. Severe HA patients (>50% of
patients) have less than 1% of normal FVIII activity, and suffer
from spontaneous haemorrhage into weight bearing joints and soft
tissues, which cause permanent disability and occasionally death.
The current standard of care for HA consists of recombinant hFVIII
protein concentrates, which can arrest haemorrhage but do not
abrogate chronic damage that ensues after a bleed. Prophylactic
administration of factor concentrates to maintain plasma FVIII
levels above 1% (>2 ng/mL) leads to a marked reduction in
spontaneous haemorrhage and chronic arthropathy. However, the half
life of FVIII is short (8-12 hours), necessitating three
intravenous administration of concentrates per week. This is
prohibitively expensive (>.English Pound.100,000/year/patient),
highly invasive and time consuming. Because of its high cost and
limited supply, over 75% of severe HA patients receive no, or only
sporadic treatment with FVIII concentrates. These individuals face
a drastically shortened life of pain and disability.
[0004] Attention has, therefore, turned to somatic gene therapy for
HA because of its potential for a cure through continuous
endogenous production of FVIII following a single administration of
vector. Haemophilia A, is in fact well suited for a gene
replacement approach because its clinical manifestations are
entirely attributable to the lack of a single gene product (FVIII)
that circulates in minute amounts (200 ng/mL) in the plasma.
Tightly regulated control of gene expression is not essential and a
modest increase in the level of FVIII (>1% of normal) can
ameliorate the severe phenotype. The availability of animal models
including FVIII-knockout mice and haemophilia A dogs can facilitate
extensive preclinical evaluation of gene therapy strategies.
Finally, the consequences of gene transfer can be assessed using
simple quantitative rather than qualitative endpoints that can be
easily assayed in most clinical laboratories, which contrasts with
other gene therapy targets where expression is difficult to assess
or is influenced by additional factors such as substrate flux.
[0005] Three gene transfer Phase I trials have been conducted thus
far for HA using direct in vivo gene delivery of onco-retro- or
adenoviral vectors as well as autologous transplant of plasmid
modified autologous fibroblasts. Stable expression of hFVIII at
above 1% was not achieved. These and subsequent preclinical studies
highlighted several critical biological obstacles to successful
gene therapy of HA.
[0006] Cellular processing of the wild type full length FVIII
molecule is highly complex and expression is confounded by mRNA
instability, interaction with endoplasmic reticulum (ER)
chaperones, and a requirement for facilitated ER to Golgi transport
through interaction with the mannose-binding lectin LMAN1. Novel
more potent FVIII variants have, however, been developed through
incremental advances in our understanding of the biology of FVIII
expression. For instance biochemical studies demonstrated that the
FVIII B-domain was dispensable for FVIII cofactor activity.
Deletion of the B-domain resulted in a 17-fold increase in mRNA
levels over full-length wild-type FVIII and a 30% increase in
secreted protein. This led to the development of B-domain deleted
(BDD) FVIII protein concentrate, which is now widely used in the
clinic. Recent studies, however, indicate that full length and BDD
hFVIII misfold in the ER lumen, resulting in activation of the
unfolded protein response (UPR) and apoptosis of murine
hepatocytes. However, the addition of a short B-domain spacer, rich
in asparagine-linked oligosaccharides, to BDD-FVIII (=N6-FVIII)
overcomes this problem in part through improved transport from the
ER to the Golgi. N6-FVIII is secreted at 10 fold higher levels than
full length wild type FVIII but the inventors believe that FVIII
secretion can be improved further through modification of the FVIII
genome.
[0007] rAAV currently shows most promise for chronic disorders such
as HA because of its excellent safety profile. In addition, the
inventors and others have shown that a single administration of
rAAV vector is sufficient to direct long-term transgene expression
without significant toxicity in a variety of animal models
including non-human primates. Integration of the rAAV provirus has
been described, but at a frequency that is exceedingly low and
comparable to that of plasmid DNA. Stable transgene expression is,
therefore, mediated mainly by episomally retained rAAV genomes in
post-mitotic tissues, thereby reducing the risk of insertional
oncogenesis. This contrasts with integrating vectors that have been
shown to cause a lymphoproliferative disorder in children with
SCID-XI. Whilst promising results have recently been reported in
patients suffering from Parkinson's disease and Leber's congenital
amaurosis following rAAV mediated gene transfer, until recently the
large size of the hFVIII cDNA (.about.7 kb), which exceeds the
relatively small packaging capacity of rAAV of .about.4.7 to 4.9
kb, has limited the use of this vector for HA. A recent report from
Pierce and colleagues demonstrated long-term (>4 years)
expression of B domain deleted (BDD) canine FVIII at 2.5-5% of
normal following a single administration of rAAV in haemophilia A
dogs. However, rAAV mediated expression of human FVIII has not been
established to the same degree.
[0008] Currently, the most severe and challenging complication of
treatment with FVIII concentrates is the development of
neutralising antibodies to FVIII (FVIII inhibitors), which occurs
in up to 30% of patients with HA. These inhibitors negate the
biological effects of FVIII concentrates and making it difficult to
treat bleeding episodes, except with bypass agents such as
recombinant activated factor VII (rFVIIa). The significant cost of
rFVIIa (.about..English Pound.500,000 per episode of orthopaedic
surgery) and toxicity (e.g. thrombosis) precludes prophylactic use.
Immune tolerance induction (ITI) is an alternative but this it is
less effective in patients with longstanding, high titre,
inhibitors. Peripheral tolerance has, in fact, been achieved in
some patients with intractable FVIII inhibitors following liver
transplantation, suggesting that stable long-term endogenous
expression of hFVIII may be important for achieving tolerance. The
inventors' data in mice and non-human primates and that of others
clearly shows that liver targeted gene transfer with rAAV promotes
a state of permanent tolerance towards the transgene through
expansion of transgene specific regulatory T cells (Tregs).
[0009] Therefore, gene transfer may provide an alternative means
for prevention and eradication of intractable inhibitors.
[0010] A key lesson from previous clinical trials with rAAV is that
preclinical studies need to be evaluated in a context relevant to
humans. They have, therefore, focused on nonhuman primates for
evaluation of rAAV vectors because, like humans, macaques are
natural hosts for AAV infection. This provides an important
opportunity to evaluate gene transfer efficiency with rAAV vectors
in out-bred animals previously sensitised to wild type AAV, which
is not possible with murine or canine models. Finally, regulatory
authorities in Europe and the United States are now requesting
preclinical safety and efficacy studies in nonhuman primates as a
condition for authorisation of a clinical trial.
[0011] To overcome the disadvantages mentioned above, the inventors
have created an improved isolated nucleotide sequence encoding
Factor VIII, along with a new promoter.
SUMMARY OF THE INVENTION
[0012] In a first aspect, the present invention provides an
isolated nucleic acid molecule comprising a nucleotide sequence
having at least 75% homology to the nucleotide sequence of SEQ ID
NO: 1 and which encodes functional factor VIII (fVIII or
FVIII).
[0013] The present invention also provides a vector comprising a
nucleic acid molecule which comprises a nucleotide sequence having
at least 75% homology to the nucleotide sequence of SEQ ID NO: 1
and which encodes functional factor VIII.
[0014] Further, the present invention provides a host cell
comprising a nucleic acid molecule as described above or a vector
as described above.
[0015] Additionally, the present invention provides a protein or
glycoprotein expressed by a host cell as described above.
[0016] Furthermore, the present invention provides a transgenic
animal comprising cells which comprise a nucleic acid molecule or a
vector as described above.
[0017] The present invention also provides a method of treating
haemophilia comprising administering a vector as described above or
a protein as described above to a patient suffering from
haemophilia.
[0018] Further, the present invention provides a nucleic acid
molecule as described above, a protein as described above or a
vector as described above for use in therapy.
[0019] Additionally, the present invention provides a nucleic acid
molecule as described above, a protein as described above or a
vector as described above for use in the treatment of
haemophilia.
[0020] Also, the present invention provides a method for delivery
of a nucleotide sequence encoding a function factor VIII to a
subject, which method comprises administering to the said subject a
nucleic acid molecule as described above, a protein as described
above or a vector as described above.
[0021] Furthermore, the present invention provides a promoter
comprising a nucleotide sequence having at least 85% homology to
the nucleotide sequence of SEQ ID NO: 3.
[0022] The present invention also provides a second vector
comprising a promoter which comprises a nucleotide sequence having
at least 85% homology to the nucleotide sequence of SEQ ID NO:
3.
[0023] Further, the present invention provides a second host cell
comprising the promoter as described above or the second vector as
described above.
[0024] Additionally, the present invention provides an expression
product expressed by the second host cell as described above,
wherein an expressible nucleotide sequence is operably linked to
the promoter.
[0025] Furthermore, the present invention provides a second
transgenic animal comprising cells which comprise the promoter as
described above or the second vector as described above.
[0026] The present invention also provides a method for the
preparation of a parvoviral gene delivery vector, the method
comprising the steps of: [0027] (a) providing an insect cell
comprising one or more nucleic acid constructs comprising: [0028]
(i) a nucleic acid molecule of any one of claims 1 to 6 that is
flanked by at least one parvoviral inverted terminal repeat
nucleotide sequence; [0029] (ii) a first expression cassette
comprising a nucleotide sequence encoding one or more parvoviral
Rep proteins which is operably linked to a promoter that is capable
of driving expression of the Rep protein(s) in the insect cell;
[0030] (iii) a second expression cassette comprising a nucleotide
sequence encoding one or more parvoviral capsid proteins which is
operably linked to a promoter that is capable of driving expression
of the capsid protein(s) in the insect cell; [0031] (b) culturing
the insect cell defined in (a) under conditions conducive to the
expression of the Rep and the capsid proteins; and, optionally,
[0032] (c) recovering the parvoviral gene delivery vector.
DETAILED DESCRIPTION OF THE INVENTION
[0033] According to a first aspect of the invention, there is
provided an isolated nucleic acid molecule comprising a nucleotide
sequence having substantial homology to the nucleotide sequence of
SEQ ID NO: 1. The term substantial homology can be further defined
with reference to a percentage homology. This is discussed in
further detail below.
[0034] The term "isolated" when used in relation to a nucleic acid
molecule of the invention typically refers to a nucleic acid
sequence that is identified and separated from at least one
contaminant nucleic acid with which it is ordinarily associated in
its natural source. Isolated nucleic acid may be present in a form
or setting that is different from that in which it is found in
nature. In contrast, non-isolated nucleic acids are nucleic acids
such as DNA and RNA found in the state they exist in nature. For
example, a given DNA sequence (e.g. a gene) is found on the host
cell chromosome in proximity to neighbouring genes; RNA sequences,
such as a specific mRNA sequence encoding a specific protein, are
found in the cell as a mixture with numerous other mRNAs which
encode a multitude of proteins. The isolated nucleic acid molecule
of the invention may be present in single-stranded or
double-stranded form. When an isolated nucleic acid molecule is to
be utilized to express a protein, it will typically contain at a
minimum the sense or coding strand (i.e., nucleic acid molecule may
be single-stranded), but may contain both the sense and anti-sense
strands (i.e., the nucleic acid molecule may be
double-stranded).
[0035] The nucleic acid molecule of the invention preferably has at
least 75%, more preferably at least 80%, more preferably still at
least 85%, even more preferably at least 90%, and more preferably
at least 95% homology, for example at least 98% homology to the
nucleotide sequence of SEQ ID NO: 1. It also preferably has at
least 70%, more preferably at least 75%, and more preferably at
least 80% homology to wild-type factor VIII. Further, the nucleic
acid molecule preferably encodes for a functional factor VIII
protein, that is to say it encodes for factor VIII which, when
expressed, has the functionality of wild type factor VIII. The
nucleic acid molecule, when expressed in a suitable system (e.g. a
host cell), produces a functional factor VIII protein and at a
relatively high level. Since the factor VIII that is produced is
functional, it will have a conformation which is the same as at
least a portion of the wild type factor VIII. In one embodiment,
the factor VIII produced by the nucleic acid will have the same
conformation as the N6 factor VIII which has been previously
described. A functional factor VIII protein produced by the
invention allows at least some blood coagulation to take place in a
subject. This causes a decrease in the time it takes for blood to
clot in a subject suffering from haemophilia. Normal factor VIII
participates in blood coagulation via the coagulation cascade.
Normal factor VIII is a cofactor for factor IXa which, in the
presence of Ca.sup.+2 and phospholipids forms a complex that
converts factor X to the activated form Xa. Therefore, a functional
factor VIII protein according to the invention can form a
functional complex with factor IXa which can convert factor X to
the activated form Xa.
[0036] Previously used factor VIII nucleotide sequences have had
problems with expression of functional protein. This is thought to
be due to inefficient expression of mRNA, protein misfolding with
subsequent intracellular degradation, and inefficient transport of
the primary translation product from the endoplasmic reticulum to
the Golgi apparatus. The inventors have found that the nucleic acid
molecule provided by the invention causes surprisingly high levels
of expression of a factor VIII protein both in vitro and in vivo.
This means that this nucleic acid molecule could be used in gene
therapy to treat haemophilia such as haemophilia A.
[0037] The nucleotide sequence of SEQ ID NO: 1 is a codon optimised
human factor VIII nucleic acid sequence which is based on the
sequence of the N6 factor VIII nucleotide sequence. The N6 factor
VIII nucleotide sequence is a Factor VIII sequence from which the B
domain has been deleted and replaced with a short B-domain spacer,
rich in asparagine-linked oligosaccharides, which improves
transport of the N6-FVIII from the ER to the Golgi.
[0038] The inventors have shown that SEQ ID NO:1 and sequences
which are similar to it, i.e. those sequences which have a
relatively high level of homology, all show surprisingly high
levels of expression of functional protein. In this regard, SEQ ID
NOs: 4, 5, 6 and 7 are also codon optimised factor VIII nucleic
acid sequences, the % homology of which are 88%, 77%, 82% and 97%
respectively, compared to SEQ ID NO: 1.
[0039] A nucleotide sequence of the invention may have at least
about 400, at least about 650, at least about 890, at least about
1140, at least about 1380, at least about 1530 of all codons coding
for the functional Factor VIII being identical to the codons (in
corresponding positions) in SEQ ID NO: 1.
[0040] The invention also provides a nucleic acid molecule which
has at least 75%, preferably at least 80%, more preferably at least
85%, even more preferably at least 90%, and more preferably at
least 95% homology, for example 98% homology to the nucleotide
sequence of SEQ ID NO: 4.
[0041] A nucleotide sequence of the invention may have at least
about 410, at least about 670, at least about 920, at least about
1180, at least about 1430, at least about 1580 of all codons coding
for the functional Factor VIII being identical to the codons (in
corresponding positions) in SEQ ID NO: 4.
[0042] The nucleotide sequence of SEQ ID NO: 4 is a codon optimised
factor VIII nucleic acid sequence which is based on the sequence of
an SQ N6 factor VIII nucleotide sequence. The SQ N6 factor VIII
nucleotide sequence is a Factor VIII sequence from which the B
domain has been deleted and replaced with an SQ link of 14 amino
acids between the a2 and a3 domains. Within the SQ link, an N6
B-domain has been inserted.
[0043] Further, the invention provides a nucleic acid molecule
which has at least 75%, preferably at least 80%, more preferably at
least 85%, even more preferably at least 90%, and more preferably
at least 95% homology, for example 98% homology to the nucleotide
sequence of SEQ ID NO: 5.
[0044] A nucleotide sequence of the invention may have at least
about 360, at least about 580, at least about 800, at least about
1020, at least about 1380, at least about 1230 of all codons coding
for the functional Factor VIII being identical to the codons (in
corresponding positions) in SEQ ID NO: 5.
[0045] The nucleotide sequence of SEQ ID NO: 5 is a codon optimised
factor VIII nucleic acid sequence which is based on the sequence of
an SQ factor VIII nucleotide sequence. The SQ factor VIII
nucleotide sequence is a Factor VIII sequence from which the B
domain has been deleted and replaced with an SQ link of 14 amino
acids between the a2 and a3 domains. The presence of the SQ link in
the complex promotes efficient intracellular cleavage of the
primary single chain translation product of 170 kDa due to the
basic arginine residues which form a recognition motif for
proteolytic cleavage by the membrane bound subtilisin-like protease
furin.
[0046] Additionally, the invention provides a nucleic acid molecule
which has at least 75%, preferably at least 80%, more preferably at
least 85%, even more preferably at least 90%, and more preferably
at least 95% homology, for example 98% homology to the nucleotide
sequence of SEQ ID NO: 6.
[0047] A nucleotide sequence of the invention may have at least
about 410, at least about 660, at least about 910, at least about
1160, at least about 1410, at least about 1560 of all codons coding
for the functional Factor VIII being identical to the codons (in
corresponding positions) in SEQ ID NO: 6.
[0048] The nucleotide sequence of SEQ ID NO: 6 is a codon optimised
factor VIII nucleic acid sequence which is based on the sequence of
an SQ Fugu B factor VIII nucleotide sequence. The SQ Fugu B factor
VIII nucleotide sequence is a Factor VIII sequence from which the B
domain has been deleted and replaced with an SQ link of 14 amino
acids between the a2 and a3 domains. Within the SQ link, a Fugu
B-domain has been inserted. A Fugu B domain is the factor VIII
B-domain from the teleost puffer fish Fugu rubripes. The Fugu B
domain has a high concentration of N-linked glycosylation sites
which greatly improve intracellular trafficking and expression of
the sequence.
[0049] Furthermore, the invention provides a nucleic acid molecule
which has at least 75%, preferably at least 80%, more preferably at
least 85%, even more preferably at least 90%, and more preferably
at least 95% homology, for example 98% homology to the nucleotide
sequence of SEQ ID NO: 7.
[0050] A nucleotide sequence of the invention may have at least
about 440, at least about 710, at least about 970, at least about
1240, at least about 1500, at least about 1670 of all codons coding
for the functional Factor VIII being identical to the codons (in
corresponding positions) in SEQ ID NO: 7.
[0051] The nucleotide sequence of SEQ ID NO: 7 is a codon optimised
factor VIII nucleic acid sequence which is based on the sequence of
the N6 factor VIII nucleotide sequence.
[0052] All the above embodiments relating to different sequences
preferably encode for a functional factor VIII. Further preferred
features are the same as those relating to SEQ ID NO: 1 where
appropriate. This will be apparent to a person skilled in the
art.
[0053] In one embodiment, any of the nucleic acid molecules of the
invention may comprise a nucleotide sequence encoding for an SQ
link in the factor VIII protein. The amino acid sequence of the SQ
link is preferably the sequence of SEQ ID NO: 18.
[0054] In another embodiment, any of the nucleic acid molecules of
the invention may comprise a nucleotide sequence encoding for an N6
B-domain in the factor VIII protein.
[0055] In a further embodiment, any of the nucleic acid molecules
of the invention may comprise a nucleotide sequence encoding for a
Fugu B-domain in the factor VIII protein.
[0056] The nucleic acid of the invention may comprise an SQ link
and an N6 B-domain in the factor VIII protein, or an SQ link and a
Fugu B-domain in the factor VIII protein.
[0057] Generally, codon optimisation does not change the amino acid
for which each codon encodes. It simply changes the nucleotide
sequence so that it is more likely to be expressed at a relatively
high level compared to the non-codon optimised sequence. Therefore,
in one embodiment, the nucleic acid molecule of the invention
encodes for a protein having between 0 and 350, between 0 and 300,
between 0 and 250, between 0 and 200, between 0 and 150, between 0
and 100, between 0 and 50, between 0 and 30, between 0 and 20,
between 0 and 15, between 0 and 10, or between 0 and 5 amino acid
changes to the protein encoded by the nucleotide sequence of SEQ ID
NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
This means that the nucleotide sequence of the nucleic acid of the
invention and, for example, SEQ ID NO: 1 (or SEQ ID NO: 4, etc.)
may be different but when they are translated the amino acid
sequence of the protein that is produced only differs by between 0
and 10 amino acids. Preferably, any amino acid changes encoded for
by the nucleic acid of the invention compared to SEQ ID NO: 1, SEQ
ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 are in the
portion of the sequence which replaced the B-domain of the factor
VIII protein, i.e. the changes do not occur in the other domains of
the protein such as the A1, a1, A2, a2, a3, A3, C1 or C2 domains.
Amino acid changes in the other domains of the factor VIII protein
affect the biological activity of the factor VIII protein.
[0058] Further, the nucleic acid molecule of the invention may
encode for a protein which is encoded by the nucleotide sequence of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID
NO: 7. This means that the nucleotide sequences of the nucleic acid
of the invention and, for example, SEQ ID NO: 1 (or SEQ ID NO: 4,
etc.) may be different but when they are translated the amino acid
sequence of the protein that is produced is the same.
[0059] In a preferred embodiment of the invention, the nucleotide
sequence coding for a functional Factor VIII has an improved codon
usage bias for the human cell as compared to naturally occurring
nucleotide sequence coding for the corresponding non-codon
optimized sequence. The adaptiveness of a nucleotide sequence
encoding a functional Factor VIII to the codon usage of human cells
may be expressed as codon adaptation index (CAI). A codon
adaptation index is herein defined as a measurement of the relative
adaptiveness of the codon usage of a gene towards the codon usage
of highly expressed human genes. The relative adaptiveness (w) of
each codon is the ratio of the usage of each codon, to that of the
most abundant codon for the same amino acid. The CAI is defined as
the geometric mean of these relative adaptiveness values.
Non-synonymous codons and termination codons (dependent on genetic
code) are excluded. CAI values range from 0 to 1, with higher
values indicating a higher proportion of the most abundant codons
(see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also
see: Kim et al., Gene. 1997, 199:293-301; zur Megede et al.,
Journal of Virology, 2000, 74: 2628-2635). Preferably, a nucleic
acid molecule encoding a Factor VIII has a CAI of at least 0.8,
0.85, 0.90, 0.92, 0.94, 0.95, 0.96 or 0.97.
[0060] In a particular embodiment, the nucleic acid molecule
encodes for a protein comprising the sequence of SEQ ID NO: 2 or
SEQ ID NO: 21 having between 0 and 250, between 0 and 200, between
0 and 150, between 0 and 100, between 0 and 50, between 0 and 30,
between 0 and 20, between 0 and 15, between 0 and 10, or between 0
and 5 amino acid changes thereto. If the nucleic acid molecule
encodes for a protein comprising the sequence of SEQ ID NO: 2 or
SEQ ID NO: 21 having changes to any of the amino acids, the protein
should still be a functional protein. A skilled person will
appreciate that minor changes can be made to some of the amino
acids of the protein without affecting the function of the protein.
Preferably, the amino acid changes are in the portion of the
sequence which replaced the B-domain of the factor VIII protein,
i.e. the changes do not occur in the other domains of the protein
such as the A1, a1, A2, a2, a3, A3, C1 or C2 domains. In other
embodiments, the nucleic acid molecule may encode for a protein
comprising or consisting of the sequence of SEQ ID NO: 2 or SEQ ID
NO: 21.
[0061] It would be well with the capabilities of a skilled person
to produce a nucleic acid molecule according to the invention. This
could be done, for example, using chemical synthesis of a given
sequence.
[0062] Further, a skilled person would readily be able to determine
whether a nucleic acid according to the invention expresses a
functional protein. Suitable methods would be apparent to those
skilled in the art. For example, one suitable in vitro method
involves inserting the nucleic acid into a vector, such as a
lentiviral or an AAV vector, transducing host cells, such as 293T
or HeLa cells, with the vector, and assaying for factor VIII
activity. Alternatively, a suitable in vivo method involves
transducing a vector containing the nucleic acid into haemophiliac
mice and assaying for functional factor VIII in the plasma of the
mice. Suitable methods are described in more detail below.
[0063] The nucleic acid can be any type of nucleic acid composed of
nucleotides. The nucleic acid should be able to be expressed so
that a protein is produced. Preferably, the nucleic acid is DNA or
RNA.
[0064] The nucleic acid molecule preferably comprises a nucleotide
sequence selected from the sequence of SEQ ID NO: 1, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. In one embodiment, the
nucleic acid molecule comprises a nucleotide sequence selected from
the sequence of SEQ ID NO: 1 and SEQ ID NO: 7. The nucleic acid
molecule may consist of a nucleotide sequence selected from the
sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6
and SEQ ID NO: 7. Further, The nucleic acid molecule may consist of
a nucleotide sequence selected from the sequence of SEQ ID NO: 1
and SEQ ID NO: 7. In one embodiment, the nucleic acid molecule
consists of a nucleotide sequence of SEQ ID NO: 1.
[0065] Also provided is a vector comprising the nucleic acid
molecule of the invention. The vector may be any appropriate
vector, including viral and non-viral vectors. Viral vectors
include lenti-, adeno-, herpes viral vectors. It is preferably a
recombinant adeno-associated viral (rAAV) vector or a lentiviral
vector. Alternatively, non-viral systems may be used, including
using naked DNA (with or without chromatin attachment regions) or
conjugated DNA that is introduced into cells by various
transfection methods such as lipids or electroporation.
[0066] The vector preferably also comprises any other components
required for expression of the nucleic acid molecule, such as
promoters. Any appropriate promoters may be used, such as LP1,
HCR-hAAT, ApoE-hAAT, and LSP. These promoters are described in more
detail in the following references: LP1: Nathwani A. et al. Blood.
2006 Apr. 1; 107(7): 2653-2661; HCR-hAAT: Miao C H et al. Mol Ther.
2000; 1: 522-532; ApoE-hAAT: Okuyama T et al. Human Gene Therapy,
7, 637-645 (1996); and LSP: Wang L et al. Proc Natl Acad Sci USA.
1999 Mar. 30; 96(7): 3906-3910.
[0067] A particular preferred promoter is provided by the
invention. Accordingly, there is provided a promoter comprising a
nucleotide sequence having substantial homology to the nucleotide
sequence of SEQ ID NO: 3. The promoter is liver specific. In one
embodiment, the nucleic acid molecule described above further
comprises a nucleotide sequence having substantial homology to the
nucleotide sequence of SEQ ID NO: 3. The term substantial homology
can be further defined with reference to a percentage homology.
This is discussed in further detail below.
[0068] A vector according to the invention may be a gene delivery
vector. Such a gene delivery vector may be a viral gene delivery
vector or a non-viral gene delivery vector.
[0069] Non-viral gene delivery may be carried out using naked DNA
which is the simplest method of non-viral transfection. It may be
possible, for example, to administer a nucleic acid of the
invention using naked plasmid DNA. Alternatively, methods such as
electroporation, sonoporation or the use of a "gene gun", which
shoots DNA coated gold particles into the cell using, for example,
high pressure gas or an inverted .22 calibre gun, may be used.
[0070] To improve the delivery of a nucleic acid into a cell, it
may be necessary to protect it from damage and its entry into the
cell may be facilitated. To this end, lipoplexes and polyplexes may
be used that have the ability to protect a nucleic acid from
undesirable degradation during the transfection process.
[0071] Plasmid DNA may be coated with lipids in an organized
structure such as a micelle or a liposome. When the organized
structure is complexed with DNA it is called a lipoplex. Anionic
and neutral lipids may be used for the construction of lipoplexes
for synthetic vectors. Preferably, however, cationic lipids, due to
their positive charge, may be used to condense negatively charged
DNA molecules so as to facilitate the encapsulation of DNA into
liposomes. If may be necessary to add helper lipids (usually
electroneutral lipids, such as DOPE) to cationic lipids so as to
form lipoplexes.
[0072] Complexes of polymers with DNA, called polyplexes, may be
used to deliver a nucleic acid of the invention. Most polyplexes
consist of cationic polymers and their production is regulated by
ionic interactions. Polyplexes typically cannot release their DNA
load into the cytoplasm. Thus, co-transfection with endosome-lytic
agents (to lyse the endosome that is made during endocytosis, the
process by which the polyplex enters the cell), such as inactivated
adenovirus, may be necessary.
[0073] Hybrid methods may be used to deliver a nucleic acid of the
invention that combines two or more techniques. Virosomes are one
example; they combine liposomes with an inactivated HIV or
influenza virus. Other methods involve mixing other viral vectors
with cationic lipids or hybridizing viruses and may be used to
deliver a nucleic acid of the invention.
[0074] A dendrimer may be used to deliver a nucleic acid of the
invention, in particular, a cationic dendrimer, i.e. one with a
positive surface charge. When in the presence of genetic material
such as DNA or RNA, charge complimentarity leads to a temporary
association of the nucleic acid with the cationic dendrimer. On
reaching its destination the dendrimer-nucleic acid complex is then
imported into the cell via endocytosis.
[0075] More typically, a suitable viral gene delivery vector may be
used to deliver a nucleic acid of the invention. Viral vectors
suitable for use in the invention may be a parvovirus, an
adenovirus, a retrovirus, a lentivirus or a herpes simplex virus.
The parvovirus may be an adenovirus-associated virus (AAV).
[0076] As used herein, in the context of gene delivery, the term
"vector" or "gene delivery vector" may refer to a particle that
functions as a gene delivery vehicle, and which comprises nucleic
acid (i.e., the vector genome) packaged within, for example, an
envelope or capsid. Alternatively, in some contexts, the term
"vector" may be used to refer only to the vector genome.
[0077] Accordingly, the present invention provides gene delivery
vectors (comprising a nucleic acid of the invention) based on
animal parvoviruses, in particular dependoviruses such as
infectious human or simian AAV, and the components thereof (e.g.,
an animal parvovirus genome) for use as vectors for introduction
and/or expression of a Factor VIII polypeptide in a mammalian cell.
The term "parvoviral" as used herein thus encompasses
dependoviruses such as any type of AAV.
[0078] Viruses of the Parvoviridae family are small DNA animal
viruses. The family Parvoviridae may be divided between two
subfamilies: the Parvovirinae, which infect vertebrates, and the
Densovirinae, which infect insects. Members of the subfamily
Parvovirinae are herein referred to as the parvoviruses and include
the genus Dependovirus. As may be deduced from the name of their
genus, members of the Dependovirus are unique in that they usually
require coinfection with a helper virus such as adenovirus or
herpes virus for productive infection in cell culture. The genus
Dependovirus includes AAV, which normally infects humans (e.g.,
serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1
and 4), and related viruses that infect other warm-blooded animals
(e.g., bovine, canine, equine, and ovine adeno-associated viruses).
Further information on parvoviruses and other members of the
Parvoviridae is described in Kenneth I. Berns, "Parvoviridae: The
Viruses and Their Replication," Chapter 69 in Fields Virology (3d
Ed. 1996). For convenience the present invention is further
exemplified and described herein by reference to AAV. It is,
however, understood that the invention is not limited to AAV but
may equally be applied to other parvoviruses.
[0079] The genomic organization of all known AAV serotypes is very
similar. The genome of AAV is a linear, single-stranded DNA
molecule that is less than about 5,000 nucleotides (nt) in length.
Inverted terminal repeats (ITRs) flank the unique coding nucleotide
sequences for the non-structural replication (Rep) proteins and the
structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the
capsid. The terminal 145 nt are self-complementary and are
organized so that an energetically stable intramolecular duplex
forming a T-shaped hairpin may be formed. These hairpin structures
function as an origin for viral DNA replication, serving as primers
for the cellular DNA polymerase complex. Following wild type (wt)
AAV infection in mammalian cells the Rep genes (i.e. encoding Rep78
and Rep52 proteins) are expressed from the P5 promoter and the P19
promoter, respectively and both Rep proteins have a function in the
replication of the viral genome. A splicing event in the Rep ORF
results in the expression of actually four Rep proteins (i.e.
Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the
unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian
cells are sufficient for AAV vector production. Also in insect
cells the Rep78 and Rep52 proteins suffice for AAV vector
production.
[0080] In an AAV suitable for use as a gene therapy vector, the
vector genome typically comprises a nucleic acid of the invention
(as described herein) to be packaged for delivery to a target cell.
According to this particular embodiment, the heterologous
nucleotide sequence is located between the viral ITRs at either end
of the vector genome. In further preferred embodiments, the
parvovirus (e. g. AAV) cap genes and parvovirus (e.g. AAV) rep
genes are deleted from the template genome (and thus from the
virion DNA produced therefrom). This configuration maximizes the
size of the nucleic acid sequence(s) that can be carried by the
parvovirus capsid.
[0081] According to this particular embodiment, the nucleic acid of
the invention is located between the viral ITRs at either end of
the substrate. It is possible for a parvoviral genome to function
with only one ITR. Thus, in a gene therapy vector of the invention
based on a parvovirus, the vector genome is flanked by at least one
ITR, but, more typically, by two AAV ITRs (generally with one
either side of the vector genome, i.e. one at the 5' end and one at
the 3' end). There may be intervening sequences between the nucleic
acid of the invention in the vector genome and one or more of the
ITRs.
[0082] Preferably, the nucleic acid encoding a functional Factor
VIII polypeptide (for expression in the mammalian cell) will be
incorporated into a parvoviral genome located between two regular
ITRs or located on either side of an ITR engineered with two D
regions.
[0083] AAV sequences that may be used in the present invention for
the production of AAV gene therapy vectors can be derived from the
genome of any AAV serotype. Generally, the AAV serotypes have
genomic sequences of significant homology at the amino acid and the
nucleic acid levels, provide an identical set of genetic functions,
produce virions which are essentially physically and functionally
equivalent, and replicate and assemble by practically identical
mechanisms. For the genomic sequence of the various AAV serotypes
and an overview of the genomic similarities see e.g. GenBank
Accession number U89790; GenBank Accession number J01901; GenBank
Accession number AF043303; GenBank Accession number AF085716;
Chiorini et al. (1997, J. Vir. 71: 6823-33); Srivastava et al.
(1983, J. Vir. 45:555-64); Chiorini et al. (1999, J. Vir.
73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu
et al. (2000, J. Vir. 74: 8635-47). AAV serotype 1, 2, 3, 4, 5, 6,
7, 8 or 9 may be used in the present invention. However, AAV
serotypes 1, 5 or 8 are preferred sources of AAV sequences for use
in the context of the present invention.
[0084] Preferably the AAV ITR sequences for use in the context of
the present invention are derived from AAV1, AAV2, AAV4 and/or
AAV6. Likewise, the Rep (Rep78 and Rep52) coding sequences are
preferably derived from AAV1, AAV2, AAV4 and/or AAV6. The sequences
coding for the VP1, VP2, and VP3 capsid proteins for use in the
context of the present invention may however be taken from any of
the known 42 serotypes, more preferably from AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like
particles obtained by e.g. capsid shuffling techniques and AAV
capsid libraries.
[0085] AAV Rep and ITR sequences are particularly conserved among
most serotypes. The Rep78 proteins of various AAV serotypes are
e.g. more than 89% identical and the total nucleotide sequence
identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6
is around 82% (Bantel-Schaal et al., 1999, J. Virol.,
73(2):939-947). Moreover, the Rep sequences and ITRs of many AAV
serotypes are known to efficiently cross-complement (i.e.,
functionally substitute) corresponding sequences from other
serotypes in production of AAV particles in mammalian cells.
US2003148506 reports that AAV Rep and ITR sequences also
efficiently cross-complement other AAV Rep and ITR sequences in
insect cells.
[0086] The AAV VP proteins are known to determine the cellular
tropicity of the AAV virion. The VP protein-encoding sequences are
significantly less conserved than Rep proteins and genes among
different AAV serotypes. The ability of Rep and ITR sequences to
cross-complement corresponding sequences of other serotypes allows
for the production of pseudotyped AAV particles comprising the
capsid proteins of a serotype (e.g., AAV1, 5 or 8) and the Rep
and/or ITR sequences of another AAV serotype (e.g., AAV2). Such
pseudotyped rAAV particles are a part of the present invention.
[0087] Modified "AAV" sequences also can be used in the context of
the present invention, e.g. for the production of AAV gene therapy
vectors. Such modified sequences e.g. include sequences having at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, or more
nucleotide and/or amino acid sequence identity (e.g., a sequence
having about 75-99% nucleotide sequence identity) to an AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP can be
used in place of wild-type AAV ITR, Rep, or VP sequences.
[0088] Although similar to other AAV serotypes in many respects,
AAV5 differs from other human and simian AAV serotypes more than
other known human and simian serotypes. In view thereof, the
production of rAAV5 can differ from production of other serotypes
in insect cells. Where methods of the invention are employed to
produce rAAV5, it is preferred that one or more constructs
comprising, collectively in the case of more than one construct, a
nucleotide sequence comprising an AAV5 ITR, a nucleotide sequence
comprises an AAV5 Rep coding sequence (i.e. a nucleotide sequence
comprises an AAV5 Rep78). Such ITR and Rep sequences can be
modified as desired to obtain efficient production of AAV5 or
pseudotyped AAV5 vectors. For example, the start codon of the Rep
sequences can be modified, VP splice sites can be modified or
eliminated, and/or the VP1 start codon and nearby nucleotides can
be modified to improve the production of AAV5 vectors.
[0089] Thus, the viral capsid used in the invention may be from any
parvovirus, either an autonomous parvovirus or dependovirus, as
described above. Preferably, the viral capsid is an AAV capsid
(e.g., AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 capsid). In general,
the AAV1 capsid or AAV6 capsid are preferred. The choice of
parvovirus capsid may be based on a number of considerations as
known in the art, e.g., the target cell type, the desired level of
expression, the nature of the heterologous nucleotide sequence to
be expressed, issues related to viral production, and the like. For
example, the AAV1 and AAV6 capsid may be advantageously employed
for skeletal muscle; AAV1, AAV5 and AAV8 for the liver and cells of
the central nervous system (e.g., brain); AAV5 for cells in the
airway and lung or brain; AAV3 for bone marrow cells; and AAV4 for
particular cells in the brain (e. g., appendable cells).
[0090] It is within the technical skills of the skilled person to
select the most appropriate virus, virus subtype or virus serotype.
Some subtypes or serotypes may be more appropriate than others for
a certain type of tissue.
[0091] For example, liver-specific expression of a nucleic acid of
the invention may advantageously be induced by AAV-mediated
transduction of liver cells. Liver is amenable to AAV-mediated
transduction, and different serotypes may be used (for example,
AAV1, AAV5 or AAV8). Transduction of muscle may be accomplished by
administration of an AAV encoding a nucleic acid of the invention
via the blood stream. Thus, intravenous or intra-arterial
administration is applicable.
[0092] A parvovirus gene therapy vector prepared according to the
invention may be a "hybrid" particle in which the viral TRs and
viral capsid are from different parvoviruses. Preferably, the viral
TRs and capsid are from different serotypes of AAV. Likewise, the
parvovirus may have a "chimeric" capsid (e. g., containing
sequences from different parvoviruses, preferably different AAV
serotypes) or a "targeted" capsid (e. g., a directed tropism).
[0093] In the context of the invention "at least one parvoviral ITR
nucleotide sequence" is understood to mean a palindromic sequence,
comprising mostly complementary, symmetrically arranged sequences
also referred to as "A," "B," and "C" regions. The ITR functions as
an origin of replication, a site having a "cis" role in
replication, i.e., being a recognition site for trans-acting
replication proteins such as e.g. Rep 78 (or Rep68) which recognize
the palindrome and specific sequences internal to the palindrome.
One exception to the symmetry of the ITR sequence is the "D" region
of the ITR. It is unique (not having a complement within one ITR).
Nicking of single-stranded DNA occurs at the junction between the A
and D regions. It is the region where new DNA synthesis initiates.
The D region normally sits to one side of the palindrome and
provides directionality to the nucleic acid replication step. A
parvovirus replicating in a mammalian cell typically has two ITR
sequences. It is, however, possible to engineer an ITR so that
binding sites are on both strands of the A regions and D regions
are located symmetrically, one on each side of the palindrome. On a
double-stranded circular DNA template (e.g., a plasmid), the Rep78-
or Rep68-assisted nucleic acid replication then proceeds in both
directions and a single ITR suffices for parvoviral replication of
a circular vector. Thus, one ITR nucleotide sequence can be used in
the context of the present invention. Preferably, however, two or
another even number of regular ITRs are used. Most preferably, two
ITR sequences are used. A preferred parvoviral ITR is an AAV ITR.
For safety reasons it may be desirable to construct a parvoviral
(AAV) vector that is unable to further propagate after initial
introduction into a cell. Such a safety mechanism for limiting
undesirable vector propagation in a recipient may be provided by
using AAV with a chimeric ITR as described in US2003148506.
[0094] Those skilled in the art will appreciate that the viral Rep
protein(s) used for producing an AAV vector of the invention may be
selected with consideration for the source of the viral ITRs. For
example, the AAV5 ITR typically interacts more efficiently with the
AAV5 Rep protein, although it is not necessary that the serotype of
ITR and Rep protein(s) are matched.
[0095] The ITR(s) used in the invention are typically functional,
i.e. they may be fully resolvable and are preferably AAV sequences,
with serotypes 1, 2, 3, 4, 5 or 6 being preferred. Resolvable AAV
ITRs according to the present invention need not have a wild-type
ITR sequence (e. g., a wild-type sequence may be altered by
insertion, deletion, truncation or missense mutations), as long as
the ITR mediates the desired functions, e. g., virus packaging,
integration, and/or provirus rescue, and the like.
[0096] Advantageously, by using a gene therapy vector as compared
with previous approaches, the restoration of protein synthesis,
i.e. factor VIII synthesis, is a characteristic that the transduced
cells acquire permanently or for a sustained period of time, thus
avoiding the need for continuous administration to achieve a
therapeutic effect.
[0097] Accordingly, the vectors of the invention therefore
represent a tool for the development of strategies for the in vivo
delivery of a nucleic acid of the invention, by engineering the
nucleic acid within a gene therapy vector that efficiently
transduces an appropriate cell type, such as a liver cell.
[0098] In a further aspect of the invention, a host is provided
comprising the vector described above. Preferably, the vector is
capable of expressing the nucleic acid molecule of the invention in
the host. The host may be any suitable host.
[0099] As used herein, the term "host" refers to organisms and/or
cells which harbour a nucleic acid molecule or a vector of the
invention, as well as organisms and/or cells that are suitable for
use in expressing a recombinant gene or protein. It is not intended
that the present invention be limited to any particular type of
cell or organism. Indeed, it is contemplated that any suitable
organism and/or cell will find use in the present invention as a
host. A host cell may be in the form of a single cell, a population
of similar or different cells, for example in the form of a culture
(such as a liquid culture or a culture on a solid substrate), an
organism or part thereof.
[0100] A host cell according to the invention may permit the
expression of a nucleic acid molecule of the invention. Thus, the
host cell may be, for example, a bacterial, a yeast, an insect or a
mammalian cell.
[0101] Any insect cell which allows for replication of a
recombinant parvoviral (rAAV) vector and which can be maintained in
culture can be used in accordance with the present invention. For
example, the cell line used can be from Spodoptera frugiperda,
drosophila cell lines, or mosquito cell lines, e.g., Aedes
albopictus derived cell lines. Preferred insect cells or cell lines
are cells from the insect species which are susceptible to
baculovirus infection, including e.g. Se301, SeIZD2109, SeUCR1,
Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5,
High Five (Invitrogen, CA, USA) and expresSF+.RTM. (U.S. Pat. No.
6,103,526; Protein Sciences Corp., CT, USA).
[0102] In addition, the invention provides a method for the
preparation of a parvoviral gene delivery vector, the method
comprising the steps of: [0103] (a) providing an insect cell
comprising one or more nucleic acid constructs comprising: [0104]
(i) a nucleic acid molecule of the invention that is flanked by at
least one parvoviral inverted terminal repeat nucleotide sequence;
[0105] (ii) a first expression cassette comprising a nucleotide
sequence encoding one or more parvoviral Rep proteins which is
operably linked to a promoter that is capable of driving expression
of the Rep protein(s) in the insect cell; [0106] (iii) a second
expression cassette comprising a nucleotide sequence encoding one
or more parvoviral capsid proteins which is operably linked to a
promoter that is capable of driving expression of the capsid
protein(s) in the insect cell; [0107] (b) culturing the insect cell
defined in (a) under conditions conducive to the expression of the
Rep and the capsid proteins; and, optionally, [0108] (c) recovering
the parvoviral gene delivery vector.
[0109] In general, therefore, the method of the invention allows
the production of a parvoviral gene delivery vector (comprising a
nucleic acid of the invention) in an insect cell. Preferably, the
method comprises the steps of: (a) culturing an insect cell as
defined above under conditions such that the parvoviral (e.g. AAV)
vector is produced; and, (b) recovering the recombinant parvoviral
(e.g. AAV) vector. Preferably, the parvoviral gene delivery vector
is an AAV gene delivery vector.
[0110] It is understood here that the (AAV) vector produced in such
a method preferably is an infectious parvoviral or AAV virion that
comprises a parvoviral genome, which itself comprises a nucleic
acid of the invention. Growing conditions for insect cells in
culture, and production of heterologous products in insect cells in
culture are well-known in the art and described e.g. in the above
cited references on molecular engineering of insects cells.
[0111] In a method of the invention, a nucleic acid of the
invention that is flanked by at least one parvoviral ITR sequence
is provided. This type of sequence is described in detail above.
Preferably, the nucleic acid of the invention is sequence is
located between two parvoviral ITR sequences.
[0112] The first expression cassette comprises a nucleotide
sequence encoding one or more parvoviral Rep proteins which is
operably linked to a first promoter that is capable of driving
expression of the Rep protein(s) in the insect cell.
[0113] A nucleotide sequence encoding animal parvoviruses Rep
proteins, is herein understood as a nucleotide sequence encoding
the non-structural Rep proteins that are required and sufficient
for parvoviral vector production in insect cells such the Rep78 and
Rep52 proteins, or the Rep68 and Rep40 proteins, or the combination
of two or more thereof.
[0114] The animal parvovirus nucleotide sequence preferably is from
a dependovirus, more preferably from a human or simian
adeno-associated virus (AAV) and most preferably from an AAV which
normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6)
or primates (e.g., serotypes 1 and 4). Rep coding sequences are
well known to those skilled in the art and suitable sequences are
referred to and described in detail in WO2007/148971 and also in
WO2009/014445.
[0115] Preferably, the nucleotide sequence encodes animal
parvoviruses Rep proteins that are required and sufficient for
parvoviral vector production in insect cells.
[0116] The second expression cassette comprises a nucleotide
sequence encoding one or more parvoviral capsid proteins which is
operably linked to a promoter that is capable of driving expression
of the capsid protein(s) in the insect cell. The capsid protein(s)
expressed may be one or more of those described above.
[0117] Preferably, the nucleotide sequence encodes animal
parvoviruses cap proteins that are required and sufficient for
parvoviral vector production in insect cells.
[0118] These three sequences (genome, rep encoding and cap
encoding) are provided in an insect cell by way of one or more
nucleic acid constructs, for example one, two or three nucleic acid
constructs. Preferably then, the one or nucleic acid constructs for
the vector genome and expression of the parvoviral Rep and cap
proteins in insect cells is an insect cell-compatible vector. An
"insect cell-compatible vector" or "vector" is understood to a
nucleic acid molecule capable of productive transformation or
transfection of an insect or insect cell. Exemplary biological
vectors include plasmids, linear nucleic acid molecules, and
recombinant viruses. Any vector can be employed as long as it is
insect cell-compatible. The vector may integrate into the insect
cells genome but the presence of the vector in the insect cell need
not be permanent and transient episomal vectors are also included.
The vectors can be introduced by any means known, for example by
chemical treatment of the cells, electroporation, or infection. In
a preferred embodiment, the vector is a baculovirus, a viral
vector, or a plasmid. In a more preferred embodiment, the vector is
a baculovirus, i.e. the construct is a baculoviral vector.
Baculoviral vectors and methods for their use are well known to
those skilled in the art.
[0119] Typically then, a method of the invention for producing a
parvoviral gene delivery vector comprises: providing to a cell
permissive for parvovirus replication (a) a nucleotide sequence
encoding a template for producing vector genome of the invention
(as described in detail herein); (b) nucleotide sequences
sufficient for replication of the template to produce a vector
genome (the first expression cassette defined above); (c)
nucleotide sequences sufficient to package the vector genome into a
parvovirus capsid (the second expression cassette defined above),
under conditions sufficient for replication and packaging of the
vector genome into the parvovirus capsid, whereby parvovirus
particles comprising the vector genome encapsidated within the
parvovirus capsid are produced in the cell. Preferably, the
parvovirus replication and/or capsid coding sequences are AAV
sequences.
[0120] A method of the invention may preferably comprise the step
of affinity-purification of the (virions comprising the)
recombinant parvoviral (rAAV) vector using an anti-AAV antibody,
preferably an immobilised antibody. The anti-AAV antibody
preferably is a monoclonal antibody. A particularly suitable
antibody is a single chain camelid antibody or a fragment thereof
as e.g. obtainable from camels or llamas (see e.g. Muyldermans,
2001, Biotechnol. 74: 277-302). The antibody for
affinity-purification of rAAV preferably is an antibody that
specifically binds an epitope on a AAV capsid protein, whereby
preferably the epitope is an epitope that is present on capsid
protein of more than one AAV serotype. E.g. the antibody may be
raised or selected on the basis of specific binding to AAV2 capsid
but at the same time also it may also specifically bind to AAV1,
AAV3, AAV5, AAV6, AAV8 or AAV9 capsids.
[0121] The invention also provides a means for delivering a nucleic
acid of the invention into a broad range of cells, including
dividing and non-dividing cells. The present invention may be
employed to deliver a nucleic acid of the invention to a cell in
vitro, e. g. to produce a polypeptide encoded by such a nucleic
acid molecule in vitro or for ex vivo gene therapy.
[0122] The nucleic acid molecule, vector, cells and methods/use of
the present invention are additionally useful in a method of
delivering a nucleic acid of the invention to a host in need
thereof, typically a host suffering from haemophilia A.
[0123] The present invention finds use in both veterinary and
medical applications. Suitable subjects for gene delivery methods
as described herein include both avians and mammals, with mammals
being preferred. The term "avian" as used herein includes, but is
not limited to, chickens, ducks, geese, quail, turkeys and
pheasants. The term "mammal" as used herein includes, but is not
limited to, humans, bovines, ovines, caprines, equines, felines,
canines, lagomorphs, etc. Human subjects are most preferred. Human
subjects include neonates, infants, juveniles, and adults.
[0124] The invention thus provides a pharmaceutical composition
comprising a nucleic acid or a vector of the invention and a
pharmaceutically acceptable carrier or diluent and/or other
medicinal agent, pharmaceutical agent or adjuvant, etc.
[0125] For injection, the carrier will typically be a liquid. For
other methods of administration, the carrier may be either solid or
liquid. For inhalation administration, the carrier will be
respirable, and will preferably be in solid or liquid particulate
form. As an injection medium, it is preferred to use water that
contains the additives usual for injection solutions, such as
stabilizing agents, salts or saline, and/or buffers.
[0126] In general, a "pharmaceutically acceptable carrier" is one
that is not toxic or unduly detrimental to cells. Exemplary
pharmaceutically acceptable carriers include sterile, pyrogen-free
water and sterile, pyrogen-free, phosphate buffered saline.
Pharmaceutically acceptable carriers include physiologically
acceptable carriers. The term "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically
compatible".
[0127] By "pharmaceutically acceptable" it is meant a material that
is not biologically or otherwise undesirable, i.e., the material
may be administered to a subject without causing any undesirable
biological effects. Thus, such a pharmaceutical composition may be
used, for example, in transfection of a cell ex vivo or in
administering a viral particle or cell directly to a subject.
[0128] A carrier may be suitable for parenteral administration,
which includes intravenous, intraperitoneal or intramuscular
administration, Alternatively, the carrier may be suitable for
sublingual or oral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
[0129] Pharmaceutical compositions are typically sterile and stable
under the conditions of manufacture and storage. Pharmaceutical
compositions may be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to accommodate high
drug concentration. The carrier may be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. In many
cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, monostearate salts
and gelatin. A nucleic acid or vector of the invention may be
administered in a time or controlled release formulation, for
example in a composition which includes a slow release polymer or
other carriers that will protect the compound against rapid
release, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers may for example be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PLG).
[0130] The parvoviral, for example AAV, vector of the invention may
be of use in transferring genetic material to a cell. Such transfer
may take place in vitro, ex vivo or in vivo.
[0131] Accordingly, the invention provides a method for delivering
a nucleotide sequence to a cell, which method comprises contacting
a nucleic acid, a vector, or a pharmaceutical composition as
described herein under conditions such the nucleic acid or vector
of the invention enters the cell. The cell may be a cell in vitro,
ex vivo or in vivo.
[0132] The invention also provides a method of treating haemophilia
comprising administering an effective amount of a nucleic acid, a
protein or a vector according to the invention to a patient
suffering from haemophilia. Preferably the patient is suffering
from haemophilia A. Preferably, the patient is human.
[0133] Further, the invention also provides a method for delivering
or administering a nucleotide sequence to a subject, which method
comprises administering to the said subject a nucleic acid, a
vector, or a pharmaceutical composition as described herein. In
particular, the present invention provides a method of
administering a nucleic acid molecule of the invention to a
subject, comprising administering to the subject a parvoviral gene
therapy vector according to the invention, optionally together with
a pharmaceutically acceptable carrier. Preferably, the parvoviral
gene therapy vector is administered in a therapeutically-effective
amount to a subject in need thereof. That is to say, administration
according to the invention is typically carried out under
conditions that result in the expression of functional Factor VIII
at a level that provides a therapeutic effect in a subject in need
thereof.
[0134] Delivery of a nucleic acid or vector of the invention to a
host cell in vivo may result in an increase of functional factor
VIII in the host, for example to a level that ameliorates one or
more symptoms of a blood clotting disorder such as haemophilia
A.
[0135] The level of naturally occurring factor VIII in a subject
suffering from haemophilia A varies depending on the severity of
the haemophilia. Patients with a severe form of the disease have
factor VIII levels of less than about 1% of the level found in a
normal healthy subject (referred to herein as "a normal level". A
normal level is about 50-150 IU/dL). Patients with a moderate form
of the disease have factor VIII levels of between about 1% and
about 5% of a normal level. Patients with a mild form of the
disease have factor VIII levels of more than about 5% of a normal
level; typically between about 5% and about 30% of a normal
level.
[0136] It has been found that when the method of treatment of the
invention is used, it can cause an increase in the level of
functional factor VIII of at least about 1% of normal levels, i.e.
in addition to the factor VIII level present in the subject before
treatment. In a subject suffering from haemophilia, such an
increase can cause amelioration of a symptom of haemophilia. In
particular, an increase of at least 1% can reduce the frequency of
bleeding that occurs in sufferers of haemophilia, especially those
with a severe form of the disease. In one embodiment, the method of
treatment causes an increase in the level of functional factor VIII
of at least about 5% of normal levels. This could change the
phenotype of the disease from severe to mild. Patients with a mild
form of the disease rarely have spontaneous bleeding. In other
embodiments, the method of treatment of the invention causes an
increase in the level of functional factor VIII of at least about
2%, at least about 3%, at least about 4%, at least about 10%, at
least about 15%, at least about 20% or at least about 25% of normal
levels. In a particular embodiment, the method of treatment of the
invention causes an increase in the level of functional factor VIII
of at least about 30% of normal levels. This level of increase
would virtually normalise coagulation of blood in subjects
suffering haemophilia. Such subjects are unlikely to require factor
VIII concentrates following trauma or during surgery.
[0137] In another embodiment, the method of treatment of the
invention may cause an increase in the level of functional factor
VIII to at least about 1% of normal levels. The method of treatment
may cause an increase in the level of functional factor VIII to at
least about 5% of normal levels. In other embodiments, the method
of treatment of the invention may cause an increase in the level of
functional factor VIII to at least about 2%, at least about 3%, at
least about 4%, at least about 10%, at least about 15%, at least
about 20% or at least about 25% of normal levels. In a particular
embodiment, the method of treatment of the invention causes an
increase in the level of functional factor VIII to at least about
30% of normal levels. A subject whose functional factor VIII level
has been increase to 30% or more will have virtually normal
coagulation of blood.
[0138] In one embodiment, the method of treatment of the invention
causes an increase in the level of functional factor VIII to, at
most, normal levels.
[0139] The level of functional factor VIII can be measured
relatively easily and methods for measuring factor VIII levels are
well known to those skilled in the art. Many clotting assays are
available, including chromogenic and clotting based assays. ELISA
tests are also widely available. A particular method is to measure
the level of factor VIII:C which is a lab measure of the clotting
activity of factor VIII. A normal level of factor VIII:C is 46.8 to
141.8 IU/dL or 0.468-1.4 IU/ml.
[0140] A further method is to measure the activated partial
thromboplastin time (aPTT) which is a measure of the ability of
blood to clot. A normal aPTT is between about 24 and about 34
seconds. A subject suffering from haemophilia will have a longer
aPTT. This method can be used in combination with prothrombin time
measurement.
[0141] Also provided is a nucleic acid molecule, protein or vector
of the invention for use in therapy, especially in the treatment of
haemophilia, particularly haemophilia A.
[0142] The use of a nucleic acid molecule, protein or vector of the
invention in the manufacture of a medicament for the treatment of
haemophilia, particularly haemophilia A, is also provided.
[0143] The invention also provides a nucleic acid or a vector of
the invention for use in the treatment of the human or animal body
by therapy. In particular, a nucleic acid or a vector of the
invention is provided for use in the treatment of a blood clotting
disorder such as haemophilia, for example haemophilia A. A nucleic
acid or a vector of the invention is provided for use in
ameliorating one or more symptoms of a blood clotting disorder, for
example by reducing the frequency and/or severity of bleeding
episodes.
[0144] The invention further provides a method of treatment of a
blood clotting disorder, which method comprises the step of
administering an effective amount of a nucleic acid or a vector of
the invention to a subject in need thereof.
[0145] Accordingly, the invention further provides use of a nucleic
acid or vector as described herein in the manufacture of a
medicament for use in the administration of a nucleic acid to a
subject. Further, the invention provides a nucleic acid or vector
as described herein in the manufacture of a medicament for use in
the treatment of a blood clotting disorder.
[0146] Typically, a nucleic acid or a vector of the invention may
be administered to a subject by gene therapy, in particular by use
of a parvoviral gene therapy vector such as AAV. General methods
for gene therapy are known in the art. The vector, composition or
pharmaceutical composition may be delivered to a cell in vitro or
ex vivo or to a subject in vivo by any suitable method known in the
art. Alternatively, the vector may be delivered to a cell ex vivo,
and the cell administered to a subject, as known in the art. In
general, the present invention can be employed to deliver any
nucleic acid of the invention to a cell in vitro, ex vivo, or in
vivo.
[0147] The present invention further provides a method of
delivering a nucleic acid to a cell. Typically, for in vitro
methods, the virus may be introduced into the cell by standard
viral transduction methods, as are known in the art.
[0148] Preferably, the virus particles are added to the cells at
the appropriate multiplicity of infection according to standard
transduction methods appropriate for the particular target cells.
Titres of virus to administer can vary, depending upon the target
cell type and the particular virus vector, and may be determined by
those of skill in the art without undue experimentation.
[0149] Cells may be removed from a subject, the parvovirus vector
is introduced therein, and the cells are then replaced back into
the subject. Methods of removing cells from subject for treatment
ex vivo, followed by introduction back into the subject are known
in the art. Alternatively, an AAV vector may be introduced into
cells from another subject, into cultured cells, or into cells from
any other suitable source, and the cells are administered to a
subject in need thereof.
[0150] A further aspect of the invention is a method of treating
subjects in vivo with a nucleic acid or vector of the invention.
Administration of a nucleic acid or vector of the present invention
to a human subject or an animal in need thereof can be by any means
known in the art for administering virus vectors.
[0151] A nucleic acid or vector of the invention will typically be
included in a pharmaceutical composition as set out above. Such
compositions include the nucleic acid or vector in an effective
amount, sufficient to provide a desired therapeutic or prophylactic
effect, and a pharmaceutically acceptable carrier or excipient. An
"effective amount" includes a therapeutically effective amount or a
prophylactically effective amount.
[0152] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, such as raising the level of
functional Factor VIII in a subject (so as to lead to functional
Factor VIII production to level sufficient to ameliorate the
symptoms of the disease associated with a lack of that
protein).
[0153] A therapeutically effective amount of a nucleic acid
molecule or vector of the invention may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the nucleic acid molecule or vector to elicit a
desired response in the individual. Dosage regimens may be adjusted
to provide the optimum therapeutic response. A therapeutically
effective amount is also typically one in which any toxic or
detrimental effects of the nucleic acid molecule or vector are
outweighed by the therapeutically beneficial effects.
[0154] Viral gene therapy vectors may be administered to a cell or
host in a biologically-effective amount. A "biologically-effective"
amount of the virus vector is an amount that is sufficient to
result in infection (or transduction) and expression of the
heterologous nucleic acid sequence in the cell. If the virus is
administered to a cell in vivo (e. g., the virus is administered to
a subject), a "biologically-effective" amount of the virus vector
is an amount that is sufficient to result in transduction and
expression of a nucleic acid according to the invention in a target
cell.
[0155] For a nucleic acid molecule or vector of the invention, such
as a gene therapy vector, the dosage to be administered may depend
to a large extent on the condition and size of the subject being
treated as well as the therapeutic formulation, frequency of
treatment and the route of administration. Regimens for continuing
therapy, including dose, formulation, and frequency may be guided
by the initial response and clinical judgment. The parenteral route
of injection into the interstitial space of tissue may be
preferred, although other parenteral routes, such as inhalation of
an aerosol formulation, may be required in specific administration.
In some protocols, a formulation comprising the gene and gene
delivery system in an aqueous carrier is injected into tissue in
appropriate amounts.
[0156] Exemplary modes of administration include oral, rectal,
transmucosal, topical, transdermal, inhalation, parenteral (e. g.,
intravenous, subcutaneous, intradermal, intramuscular, and
intraarticular) administration, and the like, as well as direct
tissue or organ injection, alternatively, intrathecal, direct
intramuscular, intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Alternatively, one may administer the
virus in a local rather than systemic manner, for example, in a
depot or sustained-release formulation.
[0157] The tissue/cell type to be administered a nucleic acid
molecule or vector of the invention may be of any type, but will
typically be a hepatic/liver cell. It is not intended that the
present invention be limited to any particular route of
administration. However, in order that liver cells are transduced,
a nucleic acid molecule or vector of the present invention may
successfully be administered via the portal or arterial
vasculature. Alternatively, the cell may be any progenitor cell. As
a further alternative, the cell can be a stem cell (e. g., a liver
stem cell). The tissue target may be specific or it may be a
combination of several tissues, for example the liver and muscle
tissues.
[0158] In the case of a gene therapy vector, the effective dose
range for small animals such as mice, following intramuscular
injection, may be between about 1.times.10.sup.11 and about
1.times.10.sup.12 genome copy (gc)/kg, and for larger animals
(cats) and possibly human subjects, between about 1.times.10.sup.10
and about 1.times.10.sup.13 gc/kg. Dosages of the parvovirus gene
therapy vector of the invention will depend upon the mode of
administration, the disease or condition to be treated, the
individual subject's condition, the particular virus vector, and
the gene to be delivered, and can be determined in a routine
manner. Typically, an amount of about 10.sup.3 to about 10.sup.16
virus particles per dose may be suitable. Preferably, an amount of
about 10.sup.9 to about 10.sup.14 virus particles per dose is used.
When treated in this way, a subject may receive a single dose of
virus particles so that the viral particles effect treatment in a
single administration.
[0159] The amount of active compound in the compositions of the
invention may vary according to factors such as the disease state,
age, sex, and weight of the individual. Dosage regimens may be
adjusted to provide the optimum therapeutic response. For example,
a single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation.
[0160] It may be advantageous to formulate parenteral compositions
in dosage unit form for ease of administration and uniformity of
dosage. "Dosage unit form" as used herein refers to physically
discrete units suited as unitary dosages for subjects to be
treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention may be
dictated by the unique characteristics of the active compound and
the particular therapeutic effect to be achieved, and by the
limitations inherent in the art of compounding such an active
compound for the treatment of a condition in individuals.
[0161] Many methods for the preparation of such formulations are
patented or generally known to those skilled in the art.
[0162] Also provided is a protein or glycoprotein expressed by a
host cell of the invention.
[0163] Further provided is a transgenic animal comprising cells
comprising a vector according to the invention. Preferably the
animal is a non-human mammal, especially a primate such as a
macaque. Alternatively, the animal may be a rodent, especially a
mouse; or may be canine, feline, ovine or porcine.
[0164] In the aspect of the invention in which a promoter is
provided comprising a nucleotide sequence having substantial
homology to the nucleotide sequence of SEQ ID NO: 3, the promoter
preferably has at least 85%, more preferably at least 90%, even
more preferably at least 95% homology to the nucleotide sequence of
SEQ ID NO: 3. The promoter is preferably less than 400 bp, more
preferably less than 350 bp, even more preferably less than 300 bp
in size.
[0165] The invention further provides a second vector comprising
the promoter of the invention. The vector may be any appropriate
vector, including viral and non-viral vectors. Viral vectors
include lenti-, adeno-, herpes viral vectors. It is preferably a
recombinant adeno-associated viral (rAAV) vector. Alternatively,
non-viral systems may be used to introduce the promoter in to a
cell, including using naked DNA (with or without chromatin
attachment regions) or conjugated DNA that is introduced into cells
by various transfection methods such as lipids or
electroporation.
[0166] The second vector may comprise any expressible nucleotide
sequence to produce an expression product, but preferably also
comprises a nucleotide sequence encoding a protein or other
molecule that should preferably be expressed in the liver,
especially a blood clotting factor. The expressible nucleotide
sequence may encode any gene that can be expressed from the liver,
including those that are not specific for liver disorders. For
instance, the liver may be used as a factory for synthesis of
interferon that is then released and systemically distributed for
the treatment of tumours at sites outside the liver. In addition to
genes, the vector can also regulate the expression of sh or siRNA.
The vector also preferably comprises any other components required
for expression of the expressible sequence.
[0167] Also provided is a second isolated nucleic acid molecule.
The isolated nucleic acid molecule comprises a first nucleotide
sequence having substantial homology to the nucleotide sequence of
SEQ ID NO: 1; and a second nucleotide sequence having substantial
homology to the nucleotide sequence of SEQ ID NO: 3. The term
substantial homology can be further defined with reference to a
percentage homology. This is discussed in further detail
herein.
[0168] In the second nucleic acid molecule (also referred to above
in the first aspect of the invention), the two sequences may be
contiguous or may be separated by a number of nucleotides. For
example, the two sequences may be separated by a kozak sequence or
one or more introns. The sequences are preferably operably linked,
that is to say the second sequence, which encodes a promoter, is
linked to the first sequence such that the first sequence may be
expressed when introduced into a cell using a vector.
[0169] Also provided is a vector comprising the second nucleic acid
molecule of the invention.
[0170] Further provided is a host cell comprising a vector
according to the invention. The host cell may be any appropriate
cell but is preferably a non-human mammalian cell, especially a
primate cell. Cells may be used to produce the protein
recombinantly, and any appropriate cell, such as a CHO cell, may be
used.
[0171] Also provided is a protein or glycoprotein expressed by a
host cell of the invention.
[0172] Further provided is a transgenic animal comprising cells
comprising a vector according to the invention. Preferably the
animal is a non-human mammal, especially a primate such as a
macaque. Alternatively, the animal may be a rodent, especially a
mouse; or may be canine, feline, ovine or porcine.
[0173] In the description above, the term "homology" is used to
refer to the similarity of two sequences. This can also be
described using the term "identity". The terms "homology" and
"identity" can be used interchangeably herein. For the purpose of
this invention, it is defined here that in order to determine the
percent identity of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first nucleic acid for optimal
alignment with a second amino or nucleic acid sequence). The
nucleotide residues at nucleotide positions are then compared. When
a position in the first sequence is occupied by the same amino acid
or nucleotide residue as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=number of identical positions/total number of positions
(i.e. overlapping positions).times.100). Preferably, the two
sequences are the same length.
[0174] A sequence comparison may be carried out over the entire
lengths of the two sequences being compared or over fragment of the
two sequences. Typically, the comparison will be carried out over
the full length of the two sequences being compared. However,
sequence identity may be carried out over a region of, for example,
about twenty, about fifty, about one hundred, about two hundred,
about five hundred, about 1000, about 2000, about 3000, about 4000,
about 4500, about 5000 or more contiguous nucleic acid
residues.
[0175] The skilled person will be aware of the fact that several
different computer programs are available to determine the homology
between two sequences. For instance, a comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. In a preferred
embodiment, the percent identity between two amino acid or nucleic
acid sequences is determined using the Needleman and Wunsch (J.
MoI. Biol. (48): 444-453 (1970)) algorithm which has been
incorporated into the GAP program in the Accelrys GCG software
package (available at http://www.accelrys.com/products/gcg/), using
either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. The skilled person will appreciate that all these different
parameters will yield slightly different results but that the
overall percentage identity of two sequences is not significantly
altered when using different algorithms.
[0176] The nucleic acid sequences of the present invention can
further be used as a "query sequence" to perform a search against
public databases to, for example, identify other family members or
related sequences. Such searches can be performed using the BLASTN
and BLASTP programs (version 2.0) of Altschul, et al. (1990) J.
Mol. Biol. 215:403-10. BLAST protein searches can be performed with
the BLASTP program, score=50, wordlength=3 to obtain amino acid
sequences homologous to protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
BLASTP and BLASTN) can be used. See the homepage of the National
Center for Biotechnology Information at
http://www.ncbi.nlm.nih.gov/.
[0177] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0178] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
[0179] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
[0180] A skilled person will appreciate that all aspects of the
invention, whether they relate to, for example, the nucleic acid,
the vector, the host cell or the use, are equally applicable to all
other aspects of the invention. In particular, aspects of the
method of treatment, for example, the administration of the nucleic
acid or vector, may have been described in greater detail than in
some of the other aspects of the invention, for example, relating
to the use of the nucleic acid or vector for treating haemophilia.
However, the skilled person will appreciate where more detailed
information has been given for a particular aspect of the
invention, this information is likely to be equally applicable to
other aspects of the invention. For example, the skilled person
will appreciate that the description relating to vectors and host
cells for the first aspect of the invention is applicable to all
vectors and host cells of the invention. Further, the skilled
person will also appreciate that the description relating to the
method of treatment is equally applicable to the use of the nucleic
acid or vector in treating haemophilia.
[0181] The invention will now be described in detail, by way of
example only, with reference to the drawings in which:
[0182] FIG. 1: Human FVIII expression in haemophilia A mice. Top
panel: A schematic of rAAV vector encoding the BDD hFVIII under the
control of the LP1 liver specific promoter. Bottom panel: Human
FVIII activity in mouse plasma at 8 weeks after tail vein
administration of 2.times.10.sup.9 vg/mouse (N=4). Naive animals
were injected with excipient.
[0183] FIG. 2: A. Human FVIII activity in mouse plasma at 30 days
following temporal vein administration of 1.times.10.sup.8 TU of
lentiviral vectors encoding either the BDD, N6 or the codop-FVIII
under the control of the SFFV promoter (N=4). B. hFVIII activity in
supernatant harvested from HepG2 cells transfected with rAAV
plasmid encoding FVIII variants under the control of the LP1
promoter or the smaller rAAV HLP-codop-FVIII expression cassette
(N=3).
[0184] FIG. 3: A. Yield of rAAV-HLP-codop-hFVIII (n=5) pseudotyped
with serotype 5 capsid when compared to the yield of scAAV-FIX
containing a self complementary 2.3 kb cassette and single stranded
rAAV vectors containing a 4.6 kb expression cassette. B. Native gel
stained with ethidium bromide and C. Alkaline gel Southern analysis
of the rAAV-HLP-codop-hFVIII viral genome derived from two separate
preparations (1 and 2).
[0185] FIG. 4: A. Mean FVIII levels.+-.SEM in murine plasma after a
single tail vein administration of rAAV-hFVIII constructs
pseudotyped with serotype 5 capsid (dose=4.times.10.sup.11
vg/mouse, N=3). B. Mean (.+-.SEM) proviral copy number in murine
liver transduced with rAAV5-hFVIII variants.
[0186] FIG. 5: A. Southern blot: Left panel showing double digest
(Kpn-1) of liver genomic DNA derived from mice (M1 and M2)
transduced with rAAV-HLP-codop-hFVIII. Right panel showing uncut
DNA or that digested with a single cutter (Not-1). HH and HT=head
to head and head to tail concantemers. B. Western blot showing a
single .about.210 kd band in the plasma of mice transduced with
rAAV-HLP-codop-hFVIII, which is not present in naive mouse plasma
or positive control consisting of full length recombinant human
FVIII (rhFVIII) diluted in mouse plasma.
[0187] FIG. 6: A. Relationship between rAAV5-HLP-codop-hFVIII dose
and hFVIII levels in murine plasma and transgene copy number at 6
weeks following gene transfer. B. Kinetics of hFVIII expression
following a single tail vein administration of 4.times.1012
vg/mouse of rAAV-HLP-codop-hFVIII pseudotyped with serotype 5 or 8
capsid. Shown are mean levels.+-.SEM.
[0188] FIG. 7: A. FVIII activity and antigen level in F8-/- mice
following a single tail vein administration of low and high dose of
rAAV-HLP-codop-hFVIII. B and C. Bleeding time and blood loss in
F8-/- mice following rAAV-HLP-codop-hFVIII gene transfer compared
to untransduced F8-/- mice and normal wild type animals.
[0189] FIG. 8: (A) Schematic representation of human FVIII variants
designed and cloned into a SIN lentiviral vector backbone. Nine
different human FVIII variants were designed and cloned into a
lentiviral vector backbone plasmid: BDD FVIII; B-domain deleted
human FVIII (4.3 kb total size). FVIII Fugu B; BDD FVIII containing
the Fugu B-domain (4.9 kb total size). FVIII N6; BDD FVIII
containing the human N6 B-domain (5.0 kb total size). SQ FVIII; BDD
FVIII containing a modified version of the SQ amino acid sequence
SQ.sup.m (4.4 kb total size). SQ FVIII Fugu B; SQ FVIII containing
the Fugu B-domain between the SQ.sup.m sequence to create the N
terminal SQ.sup.a and C terminal SQ.sup.b sequences (5.0 kb total
size). SQ FVIII N6; SQ FVIII containing the human N6 B-domain (5.1
kb total size). Constructs SQ FVIII (co) (4.4 kb total size), SQ
FVIII Fugu B (co) (5.0 kb total size), and SQ FVIII N6 (co) (5.1 kb
total size) are the same amino acid structure as constructs SQ
FVIII, SQ FVIII Fugu B, and SQ FVIII N6, respectively, but are
produced from a codon optimised cDNA sequence. Relative domain size
is not accurate. Dashes on constructs mark asparagine (N)-linked
glycosylation sites within the B-domain only. (B) Schematics of SQ
and modified SQ sequences; SQ.sup.m, SQ.sup.a and SQ.sup.b. The SQ
sequence is a 14 amino acid bridge between the a2 and a3 domains of
FVIII created by fusing Ser743 and Gln1638 in the B-domain. The
sequence promotes efficient intracellular cleavage by containing
the 4 amino acid protease recognition site RHQR. A modified SQ
sequence (SQ.sup.m) was created containing a missense mutation from
Lys1644 to Thr1644 caused by the creation of an MluI restriction
enzyme site within the cDNA sequence for insertion of the Fugu and
N6 B-domains. SQ.sup.a is the 11aa sequence created at the
N-terminal of the B-domain after insertion of the N6 or Fugu
B-domain sequences into the SQ FVIII construct. SQ.sup.b is the 5
amino acid sequence created at the C-terminal of the B-domain after
insertion of the N6 or Fugu B-domain sequences into the SQ FVIII
construct, this sequence retains the 4 amino acid protease
recognition site. MluI restriction sites are shown underlined and
the K to T missense mutation is at the left hand amino acid
position of the MluI restriction site.
[0190] FIG. 9: Relative human fVIII activity of FVIII constructs in
vitro as determined by chromogenic assay. 1.times.10.sup.5 293T
cells were transduced with serial dilutions of BDD FVIII, FVIII
Fugu B, FVIII N6, SQ FVIII, SQ FVIII Fugu B, SQ FVIII N6, SQ FVIII
(co), SQ FVIII Fugu B (co), or SQ FVIII N6 (co). At 48 hours cell
media was changed for 500 .mu.L serum free media. After a further
24 hours incubation media was collected from all wells and assayed
for factor VIII expression using a chromogenic based assay to
measure factor VIII cofactor activity. Results were then normalised
on copy number per cell determined by qPCR. Mean and SD shown for
n=5. Values above bars represent the fold increase in FVIII
expression from codon optimised constructs in comparison to
equivalent non-codon optimised sequences. * Statistical analyses
were performed using general linear model (GLM) based on two-way
analysis of variance (ANOVA) with individual pairwise comparisons
performed using Bonferroni simultaneous tests (Minitab software,
Myerstown, Pa.). Results show a highly significant increase for SQ
FVIII (co), SQ FVIII Fugu B (co), and SQ FVIII N6 (co) in
comparison to their non codon optimised equivalents SQ FVIII, SQ
FVIII Fugu B, SQ FVIII N6, respectively, (P<0.0001). In
addition, results for codon optimised vectors also show a
significant increase for SQ FVIII N6 (co) in comparison to SQ FVIII
(co) (P<0.0001), and a significant increase for SQ FVIII Fugu B
(co) in comparison to both SQ FVIII (co) and SQ FVIII N6 (co)
(P<0.0001).
[0191] FIG. 10: Expression of human FVIII activity in vivo in blood
plasma of hemophiliac mice after intravenous injection of SIN
lentiviral vectors expressing bioengineered FVIII constructs. Six
to ten F8.sup.tm2Kaz haemophilic neonatal mice were injected
intravenously via the superficial temporal vein with SIN lentiviral
vectors expressing bioengineered human FVIII constructs. Mice were
bled at various time-points over approximately 250 days and a
chromogenic assay used to calculate the activity of human FVIII in
blood plasma taken from each mouse as a percentage of normal human
levels. (A) SQ FVIII (white diamonds) vs. SQ FVIII (co) (black
triangles). (B) SQ FVIII Fugu B (white diamonds) vs. SQ FVIII Fugu
B (co) (black triangles). (C) SQ FVIII N6 (white diamonds) vs. SQ
FVIII N6 (co) (black triangles). Points on graphs represent the
mean; error bars represent the standard deviation. Statistical
analyses were performed using general linear model (GLM) based on
two-way analysis of variance (ANOVA) with individual pairwise
comparisons performed using Bonferroni simultaneous tests (Minitab
software, Myerstown, Pa.).
[0192] FIG. 11: FVIII activity levels in vivo in plasma taken from
mice injected with vector expressing FVIII from codon optimised
cDNA sequences. Activity of human FVIII in blood plasma taken from
mice injected with lentiviral vector expressing SQ FVIII (co) (grey
circles), SQ FVIII Fugu B (co) (white diamonds), and SQ FVIII N6
(co) (black triangles) collated. Points on graphs represent the
mean, error bars represent the SD. No significant difference in
expression is noted between constructs expressing different
B-domains (P>0.5, Bonferroni simultaneous test).
[0193] FIG. 12: Quantification of vector copy number in tissues of
hemophiliac mice after intravenous injection of SIN lentiviral
vectors expressing bioengineered FVIII constructs. Liver, spleen,
heart, lung and kidney tissue were taken from mice sacrificed at
.about.250 days post neonatal injection of lentiviral vectors
expressing SQ FVIII, SQ FVIII Fugu B, SQ FVIII N6, SQ FVIII (co),
SQ FVIII Fugu B (co), and SQ FVIII N6 (co). Genomic DNA was
extracted and viral copy number determined using qPCR. Line
represents the mean of all points. No significant difference in
copy number was observed between any vector group (P>0.1,
Bonferroni simultaneous test).
Example 1
[0194] Packaging of an hFVIII Expression Cassette into rAAV
[0195] The inventors have established that a 6.0 kb expression
cassette containing the BDD-FVIII cDNA under the control of the
previously described liver specific promoter (LP1) can be
efficiently packaged into rAAV vectors pseudotyped with serotype 5
capsid proteins (rAAV5-LP1-BDD-hFVIII) using the conventional 3
plasmid transient transfection method. Tail vein administration of
only 2.times.10.sup.9 rAAV5-LP1-BDD-hFVIII particles into adult
male FVIIIKO mice resulted in FVIII coagulation activity of
18.+-.5.3% using a chromogenic assay (FIG. 1), which is
significantly above the level required for amelioration of the
bleeding diathesis in humans (>1% of normal).
Scale-Up of rAAV-hFVIII Vector Production
[0196] The inventors have established a GMPP compatible, simple,
scalable rAAV production method using the baculovirus expression
vector and insect cells. A key advantage of the baculovirus system
is the ease with which production can be scaled up. It has been
possible to generate 1.times.10.sup.14 vector genomes (vg) from a
single production run using a bioreactor. This quantity would be
sufficient for a Phase I/II HA clinical trial. Initial yields with
our first generation FVIII vector (rAAV5-LP1-BDD-hFVIII) are in the
order of 5.times.10.sup.11 vg from 1 litre of cell culture.
Expression from Codon Optimised hFVIII
[0197] The inventors have designed an alternative hFVIII construct
(codop-FVIII) to test the hypothesis that replacing infrequently
used codons in the cDNA with those more commonly found in mammalian
genes ("codon optimisation") will generate increased expression of
hFVIII following gene transfer. A similar exercise for coagulation
factors IX and VII improved expression by up to 10 fold when
compared to the wild type cognates. The strategy for the design of
the codop-hFVIII involved back translating the hFVIII amino acid
sequence with a set of codons most frequently found in highly
expressed mammalian genes. This modified sequence was then
carefully scanned and codons were further modified to improve mRNA
stability and remove undesirable sequences, such as excess CpG
dinucleotides, and cryptic splice sites. The final designed
codop-hFVIII sequence contains 1076 single by changes from the wild
type N6-FVIII sequence, and is 42% A+T, relative to 56% A+T content
of the wild type sequence. The codop FVIII sequence is the sequence
of SEQ ID NO: 1. Initially, this codop-FVIII variant was cloned
into a lentiviral vector down stream of the constitutive spleen
focus-forming virus (SFFV) promoter and its potency assessed in new
born FVIIIKO mice by injecting 1.times.10.sup.8 TU into the
temporal vein. For comparison two separate cohorts of newborn
FVIIIKO mice were transduced with an equivalent titre of an
identical vector encoding either the BDD or the more potent N6
FVIII variants. As shown in FIG. 2a, and consistent with previous
reports, lentiviral vectors encoding the N6-FVIII (15.+-.0.8% of
normal) mediated 5 fold higher levels of transgene expression when
compared to the BDD variant (3.+-.0.6% of normal). In comparison,
hFVIII expression in the plasma of codop-FVIII cohort of mice
(283.+-.0.21% of normal) was at least 18 fold higher than that
achieved with N6-FVIII. The inventors have cloned the BDD, N6 and
codop FVIII variants into their standard rAAV vector (Nathwani A.
et al. Blood. 2007 Feb. 15; 109(4): 1414-1421) under the LP1
promoter. In addition, codop-FVIII has also been cloned down stream
of a new smaller hybrid liver specific promoter (HLP). The HLP
promoter has the sequence of SEQ ID NO: 3. Evaluation of these rAAV
vectors plasmids in a transient transfection assay in HepG2 liver
cell-line (FIG. 2b) showed that the LP1 rAAV vectors encoding
codop-FVIII (0.38.+-.0.06 IU/ml) mediated FVIII expression at
levels that were between 4 (0.09.+-.0.02 IU/ml) and 8 (0.05.+-.0.02
IU/ml) fold higher than achieved with rAAV-LP1-N6-FVIII and
rAAV-LP1-BDD-FVIII respectively. Collectively, therefore, these
data suggest that the inventors codop-FVIII molecule is more potent
than the N6-FVIII variant. Notably, the slightly smaller
rAAV-HLP-codop-FVIII vector plasmid consistently generates between
30-50% higher yields of vector than rAAV-LP1-codop-FVIII.
HLP-Codop-hFVIII Expression Cassette Can Be Packaged into AAV
Virions
[0198] The .about.5.6 kb rAAV-HLP-codop-hFVIII expression cassettes
exceed the 4.6 kb packaging limit of AAV vectors but was
successfully packaged into AAV virions with the same efficiency as
scAAV-FIX vector that is being used in on-going clinic trial (FIG.
3A) using the conventional HEK293T transient transfection method.
Others have shown that up to 6.6-kb vector sequence may be packaged
into AAV virions. Additionally, Dr High's group at the University
of Pennsylvania, independently verified that up to
6.times.10.sup.13 rAAV8 pseudotyped particles of
rAAV-HLP-codop-hFVIII could be derived following transient
transfection from just 20 roller-bottles of HEK293 cells (Yield
=6.times.10.sup.4 vg/293T cell). To demonstrate that the
rAAV-HLP-codop-hFVIII vector genome was packaged in its entirety,
DNA was extracted from virions derived from two separate stocks,
after DNaseI treatment and separated on native and alkaline agarose
gel and then assessed following ethidium bromide staining or
Southern blot analysis respectively. A prominent band of
approximately 5.7 kb was noted with both assessment methods (FIGS.
3B and C).
Codop-hFVIII is More Potent but as Safe as the N6 or BDD hFVIII
Variants
[0199] rAAV vectors pseudotyped with serotype 5 capsid encoding the
codop, N6 and the BDD-hFVIII variant under the control of either
the LP1 or HLP promoters were injected via the tail vein
(4.times.10.sup.11 vg/mouse, N=3/group) of male 4-6 week C57B1/6
mice. As shown in FIG. 4, a single tail vein administration of
rAAV-LP1-codop-hFVIII resulted in 0.20.+-.0.03 IU/ml (=20% of
normal levels) of hFVIII in murine plasma without any toxicity.
Expression of hFVIII was 10 fold lower in mice transduced with
4.times.10.sup.11 vg/mouse of rAAV-LP1-N6-hFVIII (0.02.+-.0.0003
IU/ml=2% of normal), which encodes the wild type hFVIII DNA
sequence instead of codon-optimised FVIII nucleotide sequence in
codop-hFVIII. This difference in expression between these two
vectors which are otherwise identical is highly significant
(p=0.0003, one way ANOVA). Replacing the LP1 promoter with the
smaller liver specific HLP promoter resulted in marginally higher
levels (0.22.+-.0.04 IU/ml) of hFVIII in the plasma of mice
transduced with 4.times.10.sup.11 vg/kg of rAAV-HLP-codop-hFVIII
when compared to the rAAV-LP1-codop-hFVIII cohort but this
difference was not significant (p=0.6). The lowest level of hFVIII
expression was observed in the plasma of mice that received
4.times.10.sup.11 vg/mouse of rAAV-LP1-BDD-hFVIII (0.01.+-.0.001
IU/ml), which approximates to 1% of normal levels. Importantly,
these differences in the level of hFVIII expression were not
related to vector copy number as qPCR analysis shows similar vector
copy number in the genomic DNA extracted from liver of animals in
each group ranging from 0.9-1 proviral copies/cell. Southern blot
analysis of genomic DNA from the liver of mice transduced with
LP1-codop-hFVIII at 6 weeks after gene transfer digested with
Kpn-1, which twice cuts within the codop-hFVIII expression
cassette, released a band of the expected size of approximately 1.9
kb (FIG. 5A). Digestion with Not-I, which is a single cutter,
generated two bands of .about.5 kb and .about.10 kb corresponding
to head-to-tail and head-to-head concatemer fragments in a ratio of
3:1 respectively. Western blot analysis showed that the
codop-hFVIII is secreted as a single chain 210 kd protein, which as
expected is smaller in size when compared to full length
recombinant FVIII (Helixate, FIG. 5B, left lane) as two thirds of
the B domain has been deleted from codop-hFVIII.
[0200] Next, different doses of rAAV5-HLP-codop-hFVIII were
administered via the tail vein to male C57B1/6 mice and plasma
hFVIII levels were assessed at 6 weeks. As shown in FIG. 6A, a
relatively linear relationship was observed between vector dose,
plasma hFVIII levels and transgene copy number with no evidence of
saturation kinetics even at the higher dose levels. Administration
of 4.times.10.sup.10 vg/mouse of rAAV5-HLP-codop-hFVIII resulted in
low but detectable hFVIII expression at 0.5% of normal. The
rAAV-HLP-codop-hFVIII transgene copy number in the liver of these
animals was also 7 fold lower (0.12.+-.0.06 copies/cell) that in
the 4.times.10.sup.11 vg/mouse dose cohort. An increase in the
vector dose to 4.times.10.sup.12 vg/mouse resulted in plasma hFVIII
levels of around 190% of physiological levels (1.9.+-.0.3 IU/ml).
The rAAV-HLP-codop-hFVIII transgene copy number in the liver of
these mice was over 330 fold higher (43.5.+-.2.5 proviral
copies/cell) than the levels observed in animals transduced with
4.times.10.sup.11 vg/mouse. and approximately in the liver. No
toxicity was observed at any of the dose levels and histological
examination of the organ after necropsy at 6 weeks did not show any
significant pathology. The transgene expression profile was next
assessed in two cohorts of mice (n=3) following tail vein
administration of 4.times.10.sup.12 vg/mouse of
rAAV-HLP-codop-hFVIII pseudotyped with serotype 5 and 8 capsid
proteins. As per previous reports by the inventors with other
single stranded rAAV vectors, hFVIII was detectable within two
weeks of gene transfer prior to reaching steady state levels of
23.+-.6 IU/ml and 54.+-.12 IU/ml by 10 weeks in mice transduced
with rAAV-HLP-codop-hFVIII pseudotyped with serotype 5 and 8 capsid
respectively (FIG. 6B). At all time points the level of hFVIII in
the rAAV8-HLP-codop-hFVIII cohort was between 2-10 fold higher when
compared to the levels achieved in mice that received serotype 5
capsid pseudotyped vector. This difference is highly significant
(p<0.001) and is consistent with similar serotype specific
differences in rAAV mediated transduction reported previously.
Plasma thrombin-antithrombin complexes (2.2.+-.0.2 .mu.g/l) were
not elevated, indicating that supraphysiological levels of hFVIII
do not induce a noticeable hypercoagulable state in mice. Finally,
anti-hFVIII antibodies were not detected in the
rAAV-HLP-codop-hFVIII mice at any stage after gene transfer.
rAAV-HLP-Codop-hFVIII Corrects Bleeding Diathesis in Haemophilia A
Mice
[0201] To confirm correction of the bleeding phenotype, the
inventors injected either 4.times.10.sup.11 (low-dose cohort, n=3)
or 5.times.10.sup.12 (high-dose cohort, n=3) rAAV5-HLP-codop-hFVIII
vector genomes into the tail vein of haemophilia A knockout mice,
which are of mixed C57B16/J-129 Sv background and contain a
deletion in exon 16 of murine FVIII. Peak hFVIII levels, as
determined by a one-stage clotting assay, were 137.+-.27% and
374.+-.18% of normal levels in the low and high-dose cohorts of
mice respectively (FIG. 7A). These levels were significantly above
background (untreated HA haemophiliac (FVIIIKO) mice hFVIII:C
level=<2% of normal) and significantly higher than the
therapeutic of >5% of normal. There was very close concordance
between hFVIII activity and antigen levels at all time points
examined with an average ratio of 1.16. The bleeding time in the
AAV treated and untreated F8-/- mice as well wild-type control mice
was assessed using a tail clip assay. The time to first arrest of
bleeding in the rAAV5-HLP-codop-hFVIII was significantly shorter
(p=0.003) at 114.+-.3 and 74.+-.14 seconds in the low and high dose
cohorts respectively when compared to untreated F8-/- mice
(311.+-.3 seconds) and comparable to that in control wild-type
animals (74.+-.20 seconds). Similarly, the amount of blood loss as
assessed by spectrophotometric analysis of the haemoglobin content
in saline into which the clipped mouse tail is immersed was
substantially lower (p=0.002) in the rAAV5-HLP-codop-hFVIII F8-/-
mice when compared to untreated F8-/- animals. Anti-hFVIII
antibodies were not detected in the rAAV treated HA mice at any
stage after gene transfer.
[0202] Collectively, therefore, these data suggest that the
codop-hFVIII molecule is more potent than the N6-hFVIII variant.
Additionally, the codop-hFVIII expression cassette appears to be
well packaged into rAAV virons despite its relatively large size
when compared to wild-type AAV genome. hFVIII is expressed as a
single chain biologically active protein following rAAV gene
transfer that is able to correct the bleeding phenotype in
haemophilia A knock out animals.
Example 2
Introduction
[0203] Hemophilia A is a serious bleeding disorder caused by a
deficiency in, or complete absence of, the blood coagulation factor
VIII (FVIII). It is the most common hereditary coagulation disorder
with an incidence approaching around 1 in 5000 males.sup.1. The
disorder is an attractive candidate for gene therapy because only a
modest increase in FVIII plasma concentration is needed for
therapeutic benefit, with levels of >1% able to achieve markedly
reduced rates of spontaneous bleeding and long term
arthropathy.sup.2. However, although preclinical results using gene
therapy in animal models of hemophilia A have been encouraging, no
approach as yet has been translated to clinical success where
insufficient levels of FVIII expression have been
observed.sup.3.
[0204] Low FVIII expression is principally caused by inefficient
expression of the mRNA.sup.4-6, a significant proportion of protein
misfolding with subsequent intracellular degradation, and
inefficient transport of the primary translation product from the
endoplasmic reticulum (ER) to the Golgi.sup.7;8. This results in
expression levels of FVIII approximately 2 to 3 orders of magnitude
lower than those of other comparably sized secreted proteins.sup.4.
Insights over the past two decades into the secretion pathway,
FVIII protein structure and function, and mechanisms of inhibitor
development have led to the incorporation of bioengineered forms of
FVIII in gene transfer systems. Bioengineering aims to improve
properties such as biosynthesis, secretion efficiency, functional
activity, plasma half-life, and to reduce
antigenicity/immunogenicity.sup.9. FVIII is produced as a large 330
kDa glycoprotein with the domain structure
A1-A2-B-A3-C1-C2.sup.10;11, where both the A and C domains have
internal sequence homology and approximately 40% sequence identity
to the A and C domains of factor V (FV), which shares the same
domain structure.sup.12;13. The B-domain, which constitutes 38% of
the total sequence, shares no amino acid sequence identity with
other known proteins, including the B-domain of FV. It is, however,
extensively glycosylated and contains 19 of the 26 asparagine
(N)-linked glycosylation sites on the whole FVIII molecule.sup.14.
FVIII B-domain is dispensable for procoagulant activity. FVIII in
which the B-domain is deleted (BDD) and replaced by a short 11
amino acid linker (FVIII SQ; FIG. 8b) is in clinical use as a
replacement recombinant FVIII product (Refacto, Wyeth
Pharma).sup.15.
[0205] It has been shown that deletion of the entire B-domain leads
to a 17-fold increase in mRNA and primary translation product,
however, only a 30% increase in the levels of secreted protein,
suggesting that the rate of ER-Golgi transport is actually
reduced.sup.16. Efficient FVIII secretion requires
carbohydrate-facilitated transport by LMAN1 (lectin mannose
binding-1) mediated by mannose residues of N-linked
oligosaccharides post-translationally attached to the B-domain. To
build on the advantages of BDD-FVIII whilst aiding LMAN1 mediated
transport Miao et al. (2004).sup.17 added back a short B-domain
sequence to the BDD-FVIII, optimally 226 amino acids and retaining
6 sites for N-linked glycosylation (226/N6). This resulted in a
10-fold increase in secretion in vitro from transfected COS-1 cells
and a 5-fold increase in vivo following hydrodynamic hepatic gene
delivery.sup.17.
[0206] The teleost puffer fish Fugu rubripes is a commonly used
organism for investigation of genetics. Fugu has a basic vertebrate
genome and contains a similar repertoire of genes to humans,
however, in 1993 it was shown that the Fugu genome is only 390 Mb,
about one-eighth the size of the human genome.sup.18. This makes
Fugu an extremely useful model for annotating the human genome and
a valuable `reference` genome for identifying genes and other
functional elements. Sequence analysis of genes in the blood
coagulation system showed that Fugu amino acid sequences are highly
conserved relative to their human orthologues. For FVIII cDNA
sequences the Fugu A1, A2, A3, C1 and C2 domains show 46, 43, 47,
52 and 50% sequence identity to human orthologues, respectively.
Conversely, the Fugu factor VIII B-domain shares only 6% sequence
identity to its human counterpart. However, although there is no
apparent sequence conservation between B-domains the Fugu B-domain
is also highly glycosylated with 11 asparagine (N)-linked
glycosylation attachment sites across its 224 amino acid
length.sup.19.
[0207] In this study the inventors examined the expression of human
BDD FVIII constructs containing the previously described `SQ`
B-domain element, the 226/N6 B-domain fragment and the Fugu
B-domain. Constructs were tested under the control of the Spleen
Focus Forming Virus (SFFV) promoter in the context of a
self-inactivating (SIN) HIV-1 based lentiviral vector (LV).
Furthermore, constructs were expressed from either a codon
optimised or non-codon optimised cDNA sequence. Multiple
transcriptional silencers and inhibitory motifs are widely
distributed throughout the FVIII cDNA.sup.4;6;20-22, and these
sequences act as potent inhibitors of RNA production and protein
formation which can hamper expression in vivo. FVIII expression for
all constructs was compared in vitro by transduction of 293T cells
and in vivo by intravenous injection of vector into neonatal
hemophilia A mice. Varying the B-domain made a significant
difference to expression of factor VIII from codon optimised cDNA
sequences in vitro, however, no difference was observed in vivo.
Direct comparison of bioengineered FVIII constructs showed that
significantly greater levels (up to a 44-fold increase and in
excess of 200% normal human levels) of active FVIII protein were
detected in the plasma of mice transduced with vector expressing
FVIII from a codon optimised cDNA sequence, successfully correcting
the disease model. To date, this is the highest relative increase
in FVIII expression following bioengineering of BDD FVIII resulting
in unprecedented, stable FVIII expression in vivo using a
lentiviral-based approach.
Methods
FVIII Transgene and Lentiviral Vector Construction
[0208] The expression plasmid pMT2-FVIII was obtained as a kind
gift from Dr. Steven W. Pipe (University of Michigan). This plasmid
contains the human FVIII gene with a Fugu B-domain. The hFVIII gene
had a B-domain deletion from amino acids 740-1649 and an MluI
restriction site (ACG'CGT) engineered by site directed mutagenesis
at amino acid positions 739-740 causing the missense mutation
Pro739 to Thr739 in the a2 domain. The Fugu B-domain had been
cloned in using flanking MluI restriction sites on 5' and 3'
creating a 4935 bp hFVIII Fugu B gene. The FVIII Fugu B gene was
removed in three parts using a digest with XhoI and KpnI to remove
a 1.83 kb fragment, a partial digest with KpnI and MluI to remove a
1.06 kb fragment, and PCR amplification of the last 2.066 kb
section using primers that created MluI and SbfI sites on the 5'
and 3' ends, respectively. Each section was sequentially cloned
into pLNT/SFFV-MCS using the same enzymes to create pLNT/SFFV-FVIII
Fugu B. The construct was fully sequenced upon completion.
pLNT/SFFV-BDD FVIII was produced by digest of pLNT/SFFV-FVIII Fugu
B with MluI to remove the Fugu B-domain and religation. The 226/N6
B-domain sequence was manufactured by GeneArt (Regensburg, Germany)
to produce a standard GeneArt plasmid containing 226/N6;
pGA_N6_nonopt, the sequence was obtained by taking the first 678 bp
of the human FVIII B-domain (cDNA found at Genbank: A05328), 5' and
3' flanking MluI sites were then added. N6 was then removed from
pGA_N6_nonopt and ligated into pLNT/SFFV-BDD FVIII using MluI to
create pLNT/SFFV-FVIII N6. The SQ cDNA sequence was obtained
from.sup.23 and was modified to contain an MluI site (underlined)
to give the SQ.sup.m cDNA sequence:
5'-AGC'TTC'AGC'CAG'AAC'CCC'CCC'GTG'CTG'ACG'CGT'CAC'CAG'CGG-3' (SEQ
ID NO: 8) (FIG. 8b). LNT/SFFV-SQ FVIII Fugu B was produced by site
directed mutagenesis performed by Eurofins MWG Operon (Ebersberg,
Germany) to add the flanking SQ.sup.a and SQ.sup.b (FIG. 8b)
sequences into the plasmid pLNT/SFFV-FVIII Fugu B to produce
pLNT/SFFV-SQ FVIII Fugu B. pLNT/SFFV-SQ FVIII was then produced by
removal of the Fugu B-domain from pLNT/SFFV-SQ FVIII Fugu B by
digest with MluI and religation. pLNT/SFFV-SQ FVIII N6 was produced
by removal of the 226/N6 B-domain from pGA_N6_nonopt by digestion
with MluI and ligation into pLNT/SFFV-SQ FVIII. In this construct
there is a repeat of the 11aa SQ.sup.a sequence caused by the
insertion of the N6 B-domain into the SQ.sup.m sequence. Codon
optimised sequences were created by analysis of the SQ FVIII Fugu B
cDNA and adaption of the codon usage to the bias of Homo sapiens
using codon adaptation index (CAI) performed by GeneArt
(Regensburg, Germany) using their in-house proprietary software
GeneOptimizer.RTM.. Optimisation also removed cis-acting sequence
motifs including internal TATA-boxes, chi-sites and ribosomal entry
sites, AT- or GC-rich sequence stretches, AU-rich elements,
inhibitory and cis-acting repressor sequence elements, repeat
sequences, RNA secondary structures, and all cryptic splice sites.
Optimisation of SQ FVIII Fugu B included the removal of 14 splice
sites, an increase in GC-content from .about.45% to .about.60% and
an increase in CAI from 0.74 to 0.97. A Kozak sequence was
introduced to increase translation initiation, and two stop codons
were added to ensure efficient termination. The optimised gene
retained the B domain flanking MluI restriction sites on the Fugu B
domain and has 75.8% sequence similarity to the original
non-optimised sequence. The optimised gene was cloned into
pLNT/SFFV-MCS to give the plasmid pLNT/SFFV-SQ FVIII Fugu B (co).
The plasmid pLNT/SFFV-SQ FVIII (co) was created by digestion of
pLNT/SFFV-SQ FVIII Fugu B (co) with MluI and religation. The 226/N6
B domain sequence from pGA_N6_nonopt was codon optimised and
manufactured by GeneArt. It was received in the plasmid pGA_N6_opt
and as the MluI restriction sites were maintained cloned directly
into the pLNT/SFFV-SQ FVIII (co) plasmid to obtain the construct
pLNT/SFFV-SQ FVIII N6 (co), again, this construct will contain an
11aa SQ.sup.a repeat sequence caused by the insertion of the B
domain into the SQ.sup.m sequence. Each construct was fully
sequenced before testing. The codon optimised SQ FVIII N6 sequence
is the sequence of SEQ ID NO: 4. The codon optimised SQ FVIII
sequence is the sequence of SEQ ID NO: 5. The codon optimised SQ
FVIII Fugu B sequence is the sequence of SEQ ID NO: 6.
Lentiviral Vector Production and Titration
[0209] Lentiviral vectors were produced by transient cotransfection
of HEK293T (293T) cells with 3 plasmids (the lentiviral vector,
pMd.G2 [vesicular stomatitis virus glycoprotein (VSV-G) envelope
plasmid], and pCMV.DELTA.8.91 [packaging plasmid, both produced by
Plasmid Factory, Bielefeld, Germany], employing polyethylenimine
(Sigma-Aldrich, Poole, UK). Viral supernatant was harvested and
concentrated using ultracentrifugation (25,000.times.g for 2 h at
4.degree. C.). Aliquots of viruses were stored at -80.degree. C.
The titres of all lentiviral vectors were determined using a
colorimetric reverse transcriptase (RT) enzyme-linked immunosorbent
assay (ELISA) kit (Roche, West Sussex, UK) according to the
manufacturer's instructions, and qPCR to determine an approximate
titre in vector genomes per mL (vg/mL).
Measurement of FVIII Activity
[0210] The cofactor activity of blood plasma samples and in vitro
cell culture media samples was assessed using the Biophen Factor
VIII:C Chromagenic Assay (Biophen, Quadratech Diagnostics, Epsom,
UK) as per manufacturer's instructions. Samples were diluted 1:20
to 1:40 in sample diluent provided and analysed in duplicate. A
standard curve in % FVIII cofactor activity was constructed by
diluting normal control plasma (Biophen, Quadratech Diagnostics)
1:20, carrying out four 1:2 serial dilutions, and running in
duplicate. Abnormal control plasma (Biophen, Quadratech
Diagnostics) was also used as a further quality control for the
assay.
Lentiviral Transduction
[0211] 293T cells were maintained in Dulbecco modified Eagle medium
(DMEM) (Gibco Life Technologies Ltd, Paisley, UK) and supplemented
with 50 IU/mL penicillin, 50 .mu.g/mL streptomycin, and 10%
heat-inactivated fetal calf serum (FCS; Gibco). For lentiviral
transduction five wells of 1.times.10.sup.5 293T cells were
transduced with serial dilutions of vector in a total volume of 300
.mu.L DMEM+10% FCS. 48 hours post-transduction cell media was
changed for 500 .mu.L OptiMEM (Gibco). After a further 24 hours
incubation media was collected from all wells and assayed for
factor VIII activity using a FVIII chromogenic assay. Genomic DNA
was then extracted from cells and viral copy number quantified
using real-time quantitative PCR (qPCR).
In Vivo Methods
[0212] All mice were handled according to procedures approved by
the UK Home Office and the Imperial College London Research Ethics
Committee. Haemophilia A mice (F8.sup.tm2Kaz) generated by deletion
of exon 17.sup.24 were maintained on a 129SV background. 0-1 day
old neonatal mice were subject to brief (<5 minutes) hypothermic
anaesthesia and 40 .mu.L of concentrated lentiviral vector
(equivalent to 4.times.10.sup.7-1.times.10.sup.8 transducing units
per mouse) injected into the superficial temporal vein. For
coagulation factor assays 100 .mu.L of peripheral blood was
collected from anaesthetised mice by tail vein bleed. Blood was
mixed immediately in a ratio of 1:9 with sodium citrate,
centrifuged at 13000 rpm in a micro-centrifuge for five minutes and
plasma transferred to fresh micro-centrifuge tube and stored at
-20.degree. C. before assaying.
Determination of Vector Copy Number by Real-Time Quantitative
PCR
[0213] Genomic DNA was extracted from cells using a standard
salting-out method.sup.25. Real-time qPCR was carried out in
triplicate for each sample to determine viral copy number. qPCR was
performed using an ABI 7000 Sequence Detection System (ABI, Applied
Biosystems, Warrington, United Kingdom). Total viral DNA was
quantified using primers 5'-TGTGTGCCCGTCTGTTGTGT-3' (SEQ ID NO: 9)
and 5'-GAGTCCTGCGTCGAGAGAGC-3' (SEQ ID NO: 10) and Taqman probe
(FAM) 5'-CGCCCGAACAGGGACTTGAA-3' (TAMRA) (SEQ ID NO: 11). The mouse
titin gene (Ttn) was used as an endogenous 2-copy gene control for
mouse cells and was quantified using primers
5'-AAAACGAGCAGTGACCTGAGG-3' (SEQ ID NO: 12) and
5'-TTCAGTCATGCTGCTAGCGC-3' (SEQ ID NO: 13) and Taqman probe (FAM)
5'-TGCACGGAATCTCGTCTCAGTC-3' (TAMRA) (SEQ ID NO: 14). The human
beta-actin gene (ACTB) was used as an endogenous 2-copy gene
control for HEK-293T cells and was quantified using primers
5'-TCACCCACAAGTTGCCCATCTACGA-3' (SEQ ID NO: 15) and
5'-CAGCGGAACCGCTCATTGCCAATGG-3' (SEQ ID NO: 16) and Taqman probe
(FAM) 5'-ATGCCCTCCCCCATGCCATCCTGCGT-3' (TAMRA) (SEQ ID NO: 17).
Statistical Analysis
[0214] Data are expressed as mean values plus or minus SD.
Statistical analyses were performed using a general linear model
(GLM) based on one-way analysis of variance (ANOVA) with individual
pairwise comparisons performed using Bonferroni simultaneous tests
(Minitab software, Myerstown, Pa.).
Results
Generation of Bioengineered FVIII Variants and Production of
FVIII-Expressing SIN Lentiviral Vectors
[0215] To overcome low protein expression associated with
haemophilia A gene transfer applications the inventors investigated
the expression from bioengineered FVIII transgenes containing
various B-domain elements from codon optimised or non-codon
optimised cDNA sequences. The following FVIII variants were
generated (FIG. 8a): BDD human FVIII containing a B-domain deletion
between amino acids 740-1649 with a missense mutation Pro739 to
Thr739 in the a2 domain previously described by Miao et al.
(2004).sup.17 (herein referred to as BDD FVIII); BDD FVIII
containing the 201aa Fugu B-domain containing 11 (N)-linked
glycosylation sites between aa's 740 and 1649 (herein referred to
as FVIII Fugu B); BDD FVIII containing the 226aa/N6 human B-domain
fragment containing 6 (N)-linked glycosylation sites between aa's
740 and 1649 previously described by Miao et al. (2004).sup.17
(herein referred to as FVIII N6); BDD FVIII containing a modified
14-amino acid SQ activation peptide SQ.sup.m between aa's 740 and
1649 (SFSQNPPVLTRHQR) (SEQ ID NO: 18) (missense mutation Lys to Thr
underlined), contains the RHQR furin recognition sequence to
increase intracellular cleavage, the original SQ activation peptide
sequence described by Sandberg et al. (2001).sup.23 (herein
referred to as SQ FVIII); SQ FVIII containing the Fugu B-domain
inserted into the SQ.sup.m sequence. This causes the SQ.sup.m
sequence to be split either side of the B-domain insert with the
N-terminal sequence (SFSQNPPVLTR) (SEQ ID NO: 19) is referred to as
SQ.sup.a, and the C-terminal sequence containing the furin
recognition site RHQR as SQ.sup.b (TRHQR) (SEQ ID NO: 20) (herein
referred to as SQ FVIII Fugu B); SQ FVIII containing the 226aa/N6
B-domain inserted into the SQ.sup.m sequence creating SQ.sup.a and
SQ.sup.b sequences on the N- and C-terminal sides of the B-domain,
respectively. In this construct there is a repeat of the 11aa
SQ.sup.a sequence caused by the insertion of the N6 B-domain into
the SQ.sup.m sequence. It is unknown the effect that this repeat
will have upon FVIII secretion and function (herein referred to as
SQ FVIII N6). Constructs `SQ FVIII (co)`, `SQ FVIII Fugu B (co)`
and `SQ FVIII N6 (co)` are identical in amino acid structure as
constructs `SQ FVIII`, `SQ FVIII Fugu B` and `SQ FVIII N6`,
respectively, but are translated from a codon optimised cDNA
sequence (FIG. 8a). Representation of SQ, SQ.sup.m, SQ.sup.a, and
SQ.sup.b are shown in FIG. 8b. All constructs were cloned into a
SIN lentiviral backbone under control of the SFFV promoter and
transgene sequences were confirmed by automated DNA sequencing.
[0216] Vectors were produced for all nine factor VIII constructs
and tested for physical titre using the reverse transcriptase
protein assay. They were then tested using qPCR to determine an
approximate titre in vector genomes per mL (vg/mL) (Table 1). There
was no substantial difference in titre between constructs.
TABLE-US-00001 TABLE 1 Physical titre of FVIII vectors as
determined by reverse transcriptase assay and qPCR. Quantification
of reverse transcriptase (RT) protein concentration in viral
stocks, measured by performing a RT colorimetric assay, quantified
in ng/.mu.L and estimated titre calculated from this. Mean shown of
n = 3. Quantification of titre in vector genomes per mL was
determined using qPCR. 1 .times. 10.sup.5 293T cells were
transduced with a serial dilution of vector, after 72 hours genomic
DNA was extracted from cells and qPCR carried out for both WPRE and
the human housekeeping gene .beta.-actin. Mean shown of n = 5.
Average Reverse Transcriptase Estimated Titre Titre Virus
(ng/.mu.L) (TU/mL) (vg/mL) BDD FVIII 10.9 3.71 .times. 10.sup.9
1.14 .times. 10.sup.8 FVIII Fugu B 46.5 1.58 .times. 10.sup.10 1.58
.times. 10.sup.9 FVIII N6 30.7 1.04 .times. 10.sup.10 1.07 .times.
10.sup.9 SQ FVIII 68.3 2.32 .times. 10.sup.10 2.91 .times. 10.sup.9
SQ FVIII Fugu B 44.8 1.52 .times. 10.sup.10 1.18 .times. 10.sup.9
SQ FVIII N6 78.0 2.65 .times. 10.sup.10 2.0 .times. 10.sup.9 SQ
FVIII (co) 69.6 2.37 .times. 10.sup.10 4.45 .times. 10.sup.9 SQ
FVIII Fugu B (co) 71.8 2.40 .times. 10.sup.10 2.65 .times. 10.sup.9
SQ FVIII N6 (co) 87.9 2.99 .times. 10.sup.10 3.39 .times.
10.sup.9
Expression of FVIII In Vitro
[0217] Relative FVIII protein expression was measured for each
construct in the human embryonic kidney cell line 293T. Cells were
transduced with a serial dilution of vector and cultured for 48
hours, after which cells were washed, fresh serum free media added
and chromogenic assays performed after a further 24 hours to
determine FVIII activity. Genomic DNA was also extracted from cells
to determine viral copy number by qPCR. Expression values were then
normalised against copy number allowing accurate values for FVIII
protein expression per gene copy to be determined (FIG. 9). All
constructs produced detectable FVIII activity using a chromogenic
assay (FIG. 9) and FVIII antigen by ELISA (data not shown).
[0218] Cells transduced with constructs expressed from non-codon
optimised cDNA sequences produced on average 1.40 to 2.89% FVIII
activity/mL/24hr/vector copy number. There was no significant
difference in expression of FVIII between equivalent constructs
where the SQ.sup.m, SQ.sup.a and SQ.sup.b activation peptide
sequences were present (P>0.05). In addition, there was no
significant increase in expression where the Fugu B or 226/N6
B-domains were present in comparison to SQ FVIII or BDD FVIII
constructs (P>0.05).
[0219] However, a highly significant increase in expression was
observed with constructs expressed from codon optimised cDNA
sequences. Cells expressing SQ FVIII (co), SQ FVIII Fugu B (co),
and SQ FVIII N6 (co) produced 22.89.+-.3.68, 47.20.+-.2.71, and
35.8.+-.2.39% FVIII activity/mL/24hr/vector copy number,
respectively, a 13- to 16-fold increase in comparison to expression
from equivalent non-codon optimised cDNA sequences (P<0.0001). A
significant increase in expression was also observed from
constructs containing the Fugu and 226/N6 B domains in comparison
to SQ FVIII (co) (P<0.0001), furthermore, the SQ FVIII Fugu B
(co) had expression significantly higher than both SQ FVIII (co)
and SQ FVIII N6 (co) (P<0.0001).
Comparison of FVIII Expression In Vivo after Intravenous Delivery
of Vector into Neonatal Haemophilia A Mice
[0220] SQ-containing FVIII expression cassettes were tested in
vivo. Six constructs; SQ FVIII, SQ FVIII Fugu B, SQ FVIII N6, SQ
FVIII (co), SQ FVIII Fugu B (co), and SQ FVIII N6 (co) were tested
by direct intravenous injection of lentiviral vector into neonatal
(0-1 day old) haemophiliac (FVIIIKO) mice. All mice received
between 4.72.times.10.sup.7 and 1.78.times.10.sup.8 vector genomes
(vg) with 6 to 10 mice injected per vector group. Blood plasma
samples were collected via tail vein bleed approximately every 30
days for a total of .about.250 days. FVIII activity was assessed
using a functional chromogenic assay.
[0221] Functional FVIII was detected in the plasma of all
transduced mice at all time points (FIG. 10). Plasma from mice
transduced with vector containing non-codon optimised FVIII
sequences; SQ FVIII, SQ FVIII Fugu B, or SQ FVIII N6 contained on
average 5.72%.+-.2.31%, 7.79%.+-.3.66%, and 9.53%.+-.2.24% normal
human FVIII activity, respectively, for the duration of the
experiment. The ability to clot rapidly following tail vein bleeds
indicated that the mice treated with sequences SQ FVIII Fugu B, or
SQ FVIII N6 were able to achieve adequate haemostasis, however 4 of
the 6 mice injected in the SQ FVIII vector group did not survive,
indicating that the levels of FVIII were insufficient to correct
the murine haemophilia A phenotype. None of the other vector groups
showed morbidity associated with low FVIII expression. For mice
transduced with vector containing codon optimised FVIII cDNA
sequences; SQ FVIII (co), SQ FVIII Fugu B (co), or SQ FVIII N6
(co), average FVIII levels were detected at 256.1%.+-.63.4%,
232.2%.+-.74.1%, and 283.7%.+-.56.2% normal human FVIII activity,
respectively, for the duration of the experiment. This is a 44-,
29-, and 29-fold increase in expression for SQ FVIII (co), SQ FVIII
Fugu B (co), and SQ FVIII N6 (co), respectively, in comparison to
expression from equivalent non-codon optimised sequences
(P<0.0001, Bonferroni simultaneous test). Furthermore, no
substantial loss in FVIII expression was observed in any vector
groups. Importantly, no significant difference in expression was
observed for constructs containing different B-domain elements for
vectors containing codon optimised or non-codon optimised cDNA
sequences (FIG. 11).
Analysis of Viral Copy Number in the Organs of Transduced Mice
[0222] From 187 and 246 days post-injection, mice were sacrificed
to determine vector copy number in liver, spleen, heart, lung and
kidney tissue by real time qPCR (FIG. 12). Vector genomes were
detected predominantly in the liver and spleen tissue with
negligible copies in heart, lung and kidney tissues for all mice in
all vector groups. Liver tissue taken from mice transduced with
vector containing non-codon optimised cDNA sequences contained an
average of 5.75, 6.97 and 5.25 vector copies per cell for SQ FVIII,
SQ FVIII Fugu B, and SQ FVIII N6, respectively. In spleen tissue
average copy number was 1.50, 3.13 and 2.75 copies per cell for SQ
FVIII, SQ FVIII Fugu B, and SQ FVIII N6, respectively. There was no
significant difference in the vector copy number detected in liver
tissues of animals injected with vector containing codon optimised
sequences (P>0.1, Bonferroni simultaneous test). Average copy
number in liver tissue was detected at 5.04, 9.17 and 8.80 copies
per cell, and in spleen tissue copy was 2.28, 2.57 and 2.60 copies
per cell, for SQ FVIII (co), SQ FVIII Fugu B (co), and SQ FVIII N6
(co), respectively. In all cases, similar copy number was found in
all tissues for all animals regardless of vector group.
Discussion
[0223] mRNA instability, interactions with resident ER chaperone
proteins, and the requirement for carbohydrate-facilitated
transport from the ER to the Golgi apparatus means that FVIII is
expressed at much lower levels from mammalian cells than other
proteins of similar size and complexity.sup.7;26. This has been a
limiting factor both in the commercial production of recombinant
FVIII for replacement therapy and in the success of gene therapy
for haemophilia A. A number of bioengineered forms of human FVIII
have been incorporated into gene transfer systems and have been
shown to have enhanced expression both in vitro and in vivo.
B-domain deleted (BDD) factor VIII constructs are used widely in
gene transfer experiments as there is no loss of FVIII procoagulant
function and its smaller size is more easily incorporated into
vectors. A variation of this construct is a BDD FVIII containing
the 14 amino acid link SQ between the A2 and A3 domains, currently
produced as a recombinant product and marketed as Refacto.TM.
(Wyeth).sup.23. The SQ link has previously been shown to promote
efficient intracellular cleavage of the primary single chain
translation product of FVIII as it contains the intracellular furin
recognition and cleavage site.sup.23;27. This construct has been
incorporated into plasmid vectors where it has conferred
therapeutic levels of expression.sup.28-30. Miao et al., in
2004.sup.17 have also shown that after plasmid transfection of
COS-1 cells a human BDD FVIII construct containing the first 226
amino acids of the B-domain including 6 N-linked asparagine
glycosylation sites was secreted 4-fold more efficiently in
comparison to BDD FVIII and 5-fold more efficiently in vivo
following hydrodynamic hepatic gene delivery.sup.17. This construct
has now been incorporated into many gene transfer vectors including
plasmid.sup.31, lentiviral vectors.sup.32, and gammaretroviral
vectors.sup.33 and is more efficiently secreted both in
vitro.sup.17;33-35 and in vivo.sup.17;35.
[0224] One of the significant limitations in the generation of
efficient viral gene delivery systems for the treatment of
hemophilia A by gene therapy is the large size of the FVIII cDNA.
The goal of this study was to investigate the effect of FVIII
expression cassettes with various B-domain constructs.
[0225] These consist of SQ FVIII, FVIII N6 and a BDD FVIII
construct containing the entire B-domain from the puffer fish Fugu
rubripes which contains 11 N-linked asparagine glycosylation sites
which potentially would promote more efficient trafficking from the
ER to the Golgi and therefore be more efficiently secreted. We also
investigated the expression of these constructs from cDNA sequences
which had been codon optimised for expression in Homo sapiens. All
constructs were tested using a SIN lentiviral vector, however, the
results are applicable to any gene delivery system. Our study found
that in vitro no difference in FVIII expression was found between
constructs with or without the modified SQ sequence. Incorporation
of B-domain regions into constructs also did not cause a
significant increase in expression for non-codon optimised
constructs in comparison to their B-domain deleted equivalents.
However, for codon optimised sequences significantly higher
expression of both SQ FVIII Fugu B (co) and SQ FVIII N6 (co) were
observed in comparison to SQ FVIII (co). A 13- to 16-fold increase
in expression of functional factor VIII per integrated gene copy
were also observed from codon optimised sequences.
[0226] In vivo, after neonatal injection of a similar number of
lentiviral vector genomes the presence of a B-domain did not
significantly affect the steady state levels of circulating FVIII
activity for either codon optimised or non-codon optimised
constructs. However, we observed a 29- to 44-fold increase in
steady state plasma levels of functional FVIII in hemophilia A mice
to levels above 200% normal human FVIII expression from codon
optimised constructs in comparison to non-codon optimised
equivalents. Importantly, these levels of circulating FVIII were
associated with a correction of the bleeding diatheses. In
contrast, the levels of FVIII activity observed in mice treated
with non-codon optimised FVIII expression cassettes were associated
with fatal haemorage following tail bleeds.
[0227] Multiple transcriptional silencers and inhibitory sequences
are widely distributed throughout the FVIII cDNA.sup.4;6;21;22 and
the increased expression following codon optimisation may be in
part due to the elimination of such sequences. However, deletion of
the entire B-domain which led to a 17-fold increase in mRNA and
primary translation product only resulted in a 30% increase in the
levels of secreted protein, suggesting that the rate of ER-Golgi
transport was reduced.sup.16 and that levels of FVIII mRNA were not
limiting expression. The introduction of multiple N-linked
glycosylation sites known to be important in ER-Golgi transport of
FVIII increased levels of secreted FVIII, suggesting that the rate
of ER-Golgi transport may be a rate limiting step.sup.17. However,
a significant amount of FVIII within the ER never transits to the
Golgi compartment due to a failure to fold correctly and misfolded
FVIII accumulation in the ER can result in oxidative damage and
apoptosis, perhaps suggesting that FVIII folding is the rate
limiting step in FVIII expression.sup.34.
[0228] Although protein secondary structure is determined primarily
by the amino acid sequence, protein folding within the cell is
affected by a range of factors: these include interaction with
other proteins (chaperones) and ligands, translocation through the
ER membrane and redox conditions. The rate of translation can also
affect protein folding and it has been suggested that codon usage
may be a mechanism to regulate translation speed and thus allow
stepwise folding of individual protein domains.sup.36;37. FVIII is
a complex multi-domain protein in which nonsequential segments of
the nascent polypeptide chain may interact in the three dimensional
fold. Ribosome stalling at `rare` codons may therefore lead to
alternative folding pathways generating altered conformations and
potentially misfolded protein. A potential explanation for the
observed effect of codon optimised sequences utilised in this study
may be that they allow efficient translation and transport across
the ER membrane allowing the nascent FVIII polypeptide chain to
fold correctly leading to the increased levels of secreted FVIII
observed in vitro and in vivo.
[0229] Expression of >200% is not required in hemophilia
patients, and production of such high levels of FVIII may be
detrimental to producer cells.sup.4;34. However, a major advantage
of the optimised sequence is the ability to minimize the number of
genetically modified cells needed to produce therapeutic levels,
thereby reducing the risk of insertional mutagenesis and insertion
site-dependent positional effects. Also, the use of strong,
ubiquitous promoter elements such as SFFV that were previously
required to drive high expression of FVIII constructs could be
replaced by weaker, tissue specific promoters which are less prone
to transcriptional silencing.sup.31.
[0230] Previous in vivo studies have demonstrated expression of
therapeutic levels of FVIII in vivo in adult haemophilia A mice
after systemic injection of vector.sup.32;38-40, transplant of
transduced bone marrow cells.sup.31;33, transplant of transduced
bone marrow cells with targeted platelet-specific
expression.sup.41;42, and transplant of transduced blood outgrowth
endothelial cells.sup.43. However, FVIII expression levels mediated
from many of these approaches have been low (1-5% normal human) and
expression transient due to formation of neutralising antibodies.
In this study we used a lentiviral gene delivery system to
investigate FVIII expression from FVIII constructs containing
various B-domains from non-codon optimised and codon optimised cDNA
sequences. We observed a dramatic increase in the level of secreted
FVIII from a codon optimised cDNA using this system, however, as
this expression cassette is only .about.5 kb in size it is
applicable for any viral (including AAV) or non-viral gene delivery
system and will allow the development of safer, more efficacious
vectors for gene therapy of haemophilia A.
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Sequence CWU 1
1
2114890DNAArtificial sequenceCodon optimised modified factor VIII
nucleotide sequence 1atg cag att gag ctg agc acc tgc ttc ttc ctg
tgc ctg ctg agg ttc 48Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu
Cys Leu Leu Arg Phe1 5 10 15tgc ttc tct gcc acc agg aga tac tac ctg
ggg gct gtg gag ctg agc 96Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu
Gly Ala Val Glu Leu Ser 20 25 30tgg gac tac atg cag tct gac ctg ggg
gag ctg cct gtg gat gcc agg 144Trp Asp Tyr Met Gln Ser Asp Leu Gly
Glu Leu Pro Val Asp Ala Arg 35 40 45ttc ccc ccc aga gtg ccc aag agc
ttc ccc ttc aac acc tct gtg gtg 192Phe Pro Pro Arg Val Pro Lys Ser
Phe Pro Phe Asn Thr Ser Val Val 50 55 60tac aag aag acc ctg ttt gtg
gag ttc act gac cac ctg ttc aac att 240Tyr Lys Lys Thr Leu Phe Val
Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80gcc aag ccc agg ccc
ccc tgg atg ggc ctg ctg ggc ccc acc atc cag 288Ala Lys Pro Arg Pro
Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95gct gag gtg tat
gac act gtg gtg atc acc ctg aag aac atg gcc agc 336Ala Glu Val Tyr
Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110cac cct
gtg agc ctg cat gct gtg ggg gtg agc tac tgg aag gcc tct 384His Pro
Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120
125gag ggg gct gag tat gat gac cag acc agc cag agg gag aag gag gat
432Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
130 135 140gac aag gtg ttc cct ggg ggc agc cac acc tat gtg tgg cag
gtg ctg 480Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln
Val Leu145 150 155 160aag gag aat ggc ccc atg gcc tct gac ccc ctg
tgc ctg acc tac agc 528Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu
Cys Leu Thr Tyr Ser 165 170 175tac ctg agc cat gtg gac ctg gtg aag
gac ctg aac tct ggc ctg att 576Tyr Leu Ser His Val Asp Leu Val Lys
Asp Leu Asn Ser Gly Leu Ile 180 185 190ggg gcc ctg ctg gtg tgc agg
gag ggc agc ctg gcc aag gag aag acc 624Gly Ala Leu Leu Val Cys Arg
Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205cag acc ctg cac aag
ttc atc ctg ctg ttt gct gtg ttt gat gag ggc 672Gln Thr Leu His Lys
Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220aag agc tgg
cac tct gaa acc aag aac agc ctg atg cag gac agg gat 720Lys Ser Trp
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235
240gct gcc tct gcc agg gcc tgg ccc aag atg cac act gtg aat ggc tat
768Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
245 250 255gtg aac agg agc ctg cct ggc ctg att ggc tgc cac agg aag
tct gtg 816Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys
Ser Val 260 265 270tac tgg cat gtg att ggc atg ggc acc acc cct gag
gtg cac agc atc 864Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu
Val His Ser Ile 275 280 285ttc ctg gag ggc cac acc ttc ctg gtc agg
aac cac agg cag gcc agc 912Phe Leu Glu Gly His Thr Phe Leu Val Arg
Asn His Arg Gln Ala Ser 290 295 300ctg gag atc agc ccc atc acc ttc
ctg act gcc cag acc ctg ctg atg 960Leu Glu Ile Ser Pro Ile Thr Phe
Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320gac ctg ggc cag ttc
ctg ctg ttc tgc cac atc agc agc cac cag cat 1008Asp Leu Gly Gln Phe
Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335gat ggc atg
gag gcc tat gtg aag gtg gac agc tgc cct gag gag ccc 1056Asp Gly Met
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350cag
ctg agg atg aag aac aat gag gag gct gag gac tat gat gat gac 1104Gln
Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360
365ctg act gac tct gag atg gat gtg gtg agg ttt gat gat gac aac agc
1152Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
370 375 380ccc agc ttc atc cag atc agg tct gtg gcc aag aag cac ccc
aag acc 1200Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro
Lys Thr385 390 395 400tgg gtg cac tac att gct gct gag gag gag gac
tgg gac tat gcc ccc 1248Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp
Trp Asp Tyr Ala Pro 405 410 415ctg gtg ctg gcc cct gat gac agg agc
tac aag agc cag tac ctg aac 1296Leu Val Leu Ala Pro Asp Asp Arg Ser
Tyr Lys Ser Gln Tyr Leu Asn 420 425 430aat ggc ccc cag agg att ggc
agg aag tac aag aag gtc agg ttc atg 1344Asn Gly Pro Gln Arg Ile Gly
Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445gcc tac act gat gaa
acc ttc aag acc agg gag gcc atc cag cat gag 1392Ala Tyr Thr Asp Glu
Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460tct ggc atc
ctg ggc ccc ctg ctg tat ggg gag gtg ggg gac acc ctg 1440Ser Gly Ile
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475
480ctg atc atc ttc aag aac cag gcc agc agg ccc tac aac atc tac ccc
1488Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495cat ggc atc act gat gtg agg ccc ctg tac agc agg agg ctg
ccc aag 1536His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu
Pro Lys 500 505 510ggg gtg aag cac ctg aag gac ttc ccc atc ctg cct
ggg gag atc ttc 1584Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro
Gly Glu Ile Phe 515 520 525aag tac aag tgg act gtg act gtg gag gat
ggc ccc acc aag tct gac 1632Lys Tyr Lys Trp Thr Val Thr Val Glu Asp
Gly Pro Thr Lys Ser Asp 530 535 540ccc agg tgc ctg acc aga tac tac
agc agc ttt gtg aac atg gag agg 1680Pro Arg Cys Leu Thr Arg Tyr Tyr
Ser Ser Phe Val Asn Met Glu Arg545 550 555 560gac ctg gcc tct ggc
ctg att ggc ccc ctg ctg atc tgc tac aag gag 1728Asp Leu Ala Ser Gly
Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575tct gtg gac
cag agg ggc aac cag atc atg tct gac aag agg aat gtg 1776Ser Val Asp
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590atc
ctg ttc tct gtg ttt gat gag aac agg agc tgg tac ctg act gag 1824Ile
Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600
605aac atc cag agg ttc ctg ccc aac cct gct ggg gtg cag ctg gag gac
1872Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
610 615 620cct gag ttc cag gcc agc aac atc atg cac agc atc aat ggc
tat gtg 1920Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly
Tyr Val625 630 635 640ttt gac agc ctg cag ctg tct gtg tgc ctg cat
gag gtg gcc tac tgg 1968Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His
Glu Val Ala Tyr Trp 645 650 655tac atc ctg agc att ggg gcc cag act
gac ttc ctg tct gtg ttc ttc 2016Tyr Ile Leu Ser Ile Gly Ala Gln Thr
Asp Phe Leu Ser Val Phe Phe 660 665 670tct ggc tac acc ttc aag cac
aag atg gtg tat gag gac acc ctg acc 2064Ser Gly Tyr Thr Phe Lys His
Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685ctg ttc ccc ttc tct
ggg gag act gtg ttc atg agc atg gag aac cct 2112Leu Phe Pro Phe Ser
Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700ggc ctg tgg
att ctg ggc tgc cac aac tct gac ttc agg aac agg ggc 2160Gly Leu Trp
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715
720atg act gcc ctg ctg aaa gtc tcc agc tgt gac aag aac act ggg gac
2208Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
725 730 735tac tat gag gac agc tat gag gac atc tct gcc tac ctg ctg
agc aag 2256Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu
Ser Lys 740 745 750aac aat gcc att gag ccc agg agc ttc agc cag aac
agc agg cac ccc 2304Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn
Ser Arg His Pro 755 760 765agc acc agg cag aag cag ttc aat gcc acc
acc atc cct gag aat gac 2352Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr
Thr Ile Pro Glu Asn Asp 770 775 780ata gag aag aca gac cca tgg ttt
gcc cac cgg acc ccc atg ccc aag 2400Ile Glu Lys Thr Asp Pro Trp Phe
Ala His Arg Thr Pro Met Pro Lys785 790 795 800atc cag aat gtg agc
agc tct gac ctg ctg atg ctg ctg agg cag agc 2448Ile Gln Asn Val Ser
Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser 805 810 815ccc acc ccc
cat ggc ctg agc ctg tct gac ctg cag gag gcc aag tat 2496Pro Thr Pro
His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830gaa
acc ttc tct gat gac ccc agc cct ggg gcc att gac agc aac aac 2544Glu
Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840
845agc ctg tct gag atg acc cac ttc agg ccc cag ctg cac cac tct ggg
2592Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly
850 855 860gac atg gtg ttc acc cct gag tct ggc ctg cag ctg agg ctg
aat gag 2640Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu
Asn Glu865 870 875 880aag ctg ggc acc act gct gcc act gag ctg aag
aag ctg gac ttc aaa 2688Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys
Lys Leu Asp Phe Lys 885 890 895gtc tcc agc acc agc aac aac ctg atc
agc acc atc ccc tct gac aac 2736Val Ser Ser Thr Ser Asn Asn Leu Ile
Ser Thr Ile Pro Ser Asp Asn 900 905 910ctg gct gct ggc act gac aac
acc agc agc ctg ggc ccc ccc agc atg 2784Leu Ala Ala Gly Thr Asp Asn
Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925cct gtg cac tat gac
agc cag ctg gac acc acc ctg ttt ggc aag aag 2832Pro Val His Tyr Asp
Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940agc agc ccc
ctg act gag tct ggg ggc ccc ctg agc ctg tct gag gag 2880Ser Ser Pro
Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu945 950 955
960aac aat gac agc aag ctg ctg gag tct ggc ctg atg aac agc cag gag
2928Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu
965 970 975agc agc tgg ggc aag aat gtg agc acc agg agc ttc cag aag
aag acc 2976Ser Ser Trp Gly Lys Asn Val Ser Thr Arg Ser Phe Gln Lys
Lys Thr 980 985 990agg cac tac ttc att gct gct gtg gag agg ctg tgg
gac tat ggc atg 3024Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp
Asp Tyr Gly Met 995 1000 1005agc agc agc ccc cat gtg ctg agg aac
agg gcc cag tct ggc tct 3069Ser Ser Ser Pro His Val Leu Arg Asn Arg
Ala Gln Ser Gly Ser 1010 1015 1020gtg ccc cag ttc aag aag gtg gtg
ttc cag gag ttc act gat ggc 3114Val Pro Gln Phe Lys Lys Val Val Phe
Gln Glu Phe Thr Asp Gly 1025 1030 1035agc ttc acc cag ccc ctg tac
aga ggg gag ctg aat gag cac ctg 3159Ser Phe Thr Gln Pro Leu Tyr Arg
Gly Glu Leu Asn Glu His Leu 1040 1045 1050ggc ctg ctg ggc ccc tac
atc agg gct gag gtg gag gac aac atc 3204Gly Leu Leu Gly Pro Tyr Ile
Arg Ala Glu Val Glu Asp Asn Ile 1055 1060 1065atg gtg acc ttc agg
aac cag gcc agc agg ccc tac agc ttc tac 3249Met Val Thr Phe Arg Asn
Gln Ala Ser Arg Pro Tyr Ser Phe Tyr 1070 1075 1080agc agc ctg atc
agc tat gag gag gac cag agg cag ggg gct gag 3294Ser Ser Leu Ile Ser
Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu 1085 1090 1095ccc agg aag
aac ttt gtg aag ccc aat gaa acc aag acc tac ttc 3339Pro Arg Lys Asn
Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe 1100 1105 1110tgg aag
gtg cag cac cac atg gcc ccc acc aag gat gag ttt gac 3384Trp Lys Val
Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp 1115 1120 1125tgc
aag gcc tgg gcc tac ttc tct gat gtg gac ctg gag aag gat 3429Cys Lys
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp 1130 1135
1140gtg cac tct ggc ctg att ggc ccc ctg ctg gtg tgc cac acc aac
3474Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn
1145 1150 1155acc ctg aac cct gcc cat ggc agg cag gtg act gtg cag
gag ttt 3519Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu
Phe 1160 1165 1170gcc ctg ttc ttc acc atc ttt gat gaa acc aag agc
tgg tac ttc 3564Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp
Tyr Phe 1175 1180 1185act gag aac atg gag agg aac tgc agg gcc ccc
tgc aac atc cag 3609Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys
Asn Ile Gln 1190 1195 1200atg gag gac ccc acc ttc aag gag aac tac
agg ttc cat gcc atc 3654Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg
Phe His Ala Ile 1205 1210 1215aat ggc tac atc atg gac acc ctg cct
ggc ctg gtg atg gcc cag 3699Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly
Leu Val Met Ala Gln 1220 1225 1230gac cag agg atc agg tgg tac ctg
ctg agc atg ggc agc aat gag 3744Asp Gln Arg Ile Arg Trp Tyr Leu Leu
Ser Met Gly Ser Asn Glu 1235 1240 1245aac atc cac agc atc cac ttc
tct ggc cat gtg ttc act gtg agg 3789Asn Ile His Ser Ile His Phe Ser
Gly His Val Phe Thr Val Arg 1250 1255 1260aag aag gag gag tac aag
atg gcc ctg tac aac ctg tac cct ggg 3834Lys Lys Glu Glu Tyr Lys Met
Ala Leu Tyr Asn Leu Tyr Pro Gly 1265 1270 1275gtg ttt gag act gtg
gag atg ctg ccc agc aag gct ggc atc tgg 3879Val Phe Glu Thr Val Glu
Met Leu Pro Ser Lys Ala Gly Ile Trp 1280 1285 1290agg gtg gag tgc
ctg att ggg gag cac ctg cat gct ggc atg agc 3924Arg Val Glu Cys Leu
Ile Gly Glu His Leu His Ala Gly Met Ser 1295 1300 1305acc ctg ttc
ctg gtg tac agc aac aag tgc cag acc ccc ctg ggc 3969Thr Leu Phe Leu
Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly 1310 1315 1320atg gcc
tct ggc cac atc agg gac ttc cag atc act gcc tct ggc 4014Met Ala Ser
Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly 1325 1330 1335cag
tat ggc cag tgg gcc ccc aag ctg gcc agg ctg cac tac tct 4059Gln Tyr
Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser 1340 1345
1350ggc agc atc aat gcc tgg agc acc aag gag ccc ttc agc tgg atc
4104Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile
1355 1360 1365aag gtg gac ctg ctg gcc ccc atg atc atc cat ggc atc
aag acc 4149Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys
Thr 1370 1375 1380cag ggg gcc agg cag aag ttc agc agc ctg tac atc
agc cag ttc 4194Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser
Gln Phe 1385 1390 1395atc atc atg tac agc ctg gat ggc aag aag tgg
cag acc tac agg 4239Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln
Thr Tyr Arg 1400 1405 1410ggc aac agc act ggc acc ctg atg gtg ttc
ttt ggc aat gtg gac 4284Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe
Gly Asn Val Asp 1415 1420 1425agc tct ggc atc aag cac aac atc ttc
aac ccc ccc atc att gcc 4329Ser Ser Gly Ile Lys His Asn Ile Phe Asn
Pro Pro Ile Ile Ala 1430 1435 1440aga tac atc agg ctg cac ccc acc
cac tac agc atc agg agc acc 4374Arg Tyr Ile Arg Leu His Pro Thr His
Tyr Ser Ile Arg Ser Thr 1445 1450 1455ctg agg atg gag ctg atg ggc
tgt gac ctg aac agc tgc agc atg 4419Leu Arg Met Glu Leu Met Gly Cys
Asp Leu Asn Ser Cys Ser Met 1460 1465 1470ccc ctg ggc atg gag agc
aag gcc atc tct gat gcc cag atc act 4464Pro Leu Gly Met Glu Ser Lys
Ala Ile Ser Asp Ala Gln Ile Thr 1475 1480 1485gcc agc agc tac ttc
acc aac atg ttt gcc acc tgg agc ccc agc 4509Ala Ser Ser Tyr Phe Thr
Asn Met Phe Ala Thr Trp Ser Pro Ser 1490 1495 1500aag gcc agg ctg
cac ctg cag ggc agg agc aat gcc tgg agg ccc 4554Lys Ala Arg Leu His
Leu Gln Gly Arg Ser Asn Ala Trp
Arg Pro 1505 1510 1515cag gtc aac aac ccc aag gag tgg ctg cag gtg
gac ttc cag aag 4599Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
Phe Gln Lys 1520 1525 1530acc atg aag gtg act ggg gtg acc acc cag
ggg gtg aag agc ctg 4644Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly
Val Lys Ser Leu 1535 1540 1545ctg acc agc atg tat gtg aag gag ttc
ctg atc agc agc agc cag 4689Leu Thr Ser Met Tyr Val Lys Glu Phe Leu
Ile Ser Ser Ser Gln 1550 1555 1560gat ggc cac cag tgg acc ctg ttc
ttc cag aat ggc aag gtg aag 4734Asp Gly His Gln Trp Thr Leu Phe Phe
Gln Asn Gly Lys Val Lys 1565 1570 1575gtg ttc cag ggc aac cag gac
agc ttc acc cct gtg gtg aac agc 4779Val Phe Gln Gly Asn Gln Asp Ser
Phe Thr Pro Val Val Asn Ser 1580 1585 1590ctg gac ccc ccc ctg ctg
acc aga tac ctg agg att cac ccc cag 4824Leu Asp Pro Pro Leu Leu Thr
Arg Tyr Leu Arg Ile His Pro Gln 1595 1600 1605agc tgg gtg cac cag
att gcc ctg agg atg gag gtg ctg ggc tgt 4869Ser Trp Val His Gln Ile
Ala Leu Arg Met Glu Val Leu Gly Cys 1610 1615 1620gag gcc cag gac
ctg tac tga 4890Glu Ala Gln Asp Leu Tyr 162521629PRTArtificial
sequenceModified Factor VIII protein 2Met Gln Ile Glu Leu Ser Thr
Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg
Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln
Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg
Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys
Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala
Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90
95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys
Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg
Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr
Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala
Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val
Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln
Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215
220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg
Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr
Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly
Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly
Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr
Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser
Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp
Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330
335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp
Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp
Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala
Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala
Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro
Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala
Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455
460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr
Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr
Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr
Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe
Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val
Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu
Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp
Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570
575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu
Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val
Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His
Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser
Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile
Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu
Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695
700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg
Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys
Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser
Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser
Phe Ser Gln Asn Ser Arg His Pro 755 760 765Ser Thr Arg Gln Lys Gln
Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp 770 775 780Ile Glu Lys Thr
Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys785 790 795 800Ile
Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser 805 810
815Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr
820 825 830Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser
Asn Asn 835 840 845Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu
His His Ser Gly 850 855 860Asp Met Val Phe Thr Pro Glu Ser Gly Leu
Gln Leu Arg Leu Asn Glu865 870 875 880Lys Leu Gly Thr Thr Ala Ala
Thr Glu Leu Lys Lys Leu Asp Phe Lys 885 890 895Val Ser Ser Thr Ser
Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn 900 905 910Leu Ala Ala
Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925Pro
Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935
940Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu
Glu945 950 955 960Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met
Asn Ser Gln Glu 965 970 975Ser Ser Trp Gly Lys Asn Val Ser Thr Arg
Ser Phe Gln Lys Lys Thr 980 985 990Arg His Tyr Phe Ile Ala Ala Val
Glu Arg Leu Trp Asp Tyr Gly Met 995 1000 1005Ser Ser Ser Pro His
Val Leu Arg Asn Arg Ala Gln Ser Gly Ser 1010 1015 1020Val Pro Gln
Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly 1025 1030 1035Ser
Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu 1040 1045
1050Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile
1055 1060 1065Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser
Phe Tyr 1070 1075 1080Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg
Gln Gly Ala Glu 1085 1090 1095Pro Arg Lys Asn Phe Val Lys Pro Asn
Glu Thr Lys Thr Tyr Phe 1100 1105 1110Trp Lys Val Gln His His Met
Ala Pro Thr Lys Asp Glu Phe Asp 1115 1120 1125Cys Lys Ala Trp Ala
Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp 1130 1135 1140Val His Ser
Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn 1145 1150 1155Thr
Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe 1160 1165
1170Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe
1175 1180 1185Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
Ile Gln 1190 1195 1200Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg
Phe His Ala Ile 1205 1210 1215Asn Gly Tyr Ile Met Asp Thr Leu Pro
Gly Leu Val Met Ala Gln 1220 1225 1230Asp Gln Arg Ile Arg Trp Tyr
Leu Leu Ser Met Gly Ser Asn Glu 1235 1240 1245Asn Ile His Ser Ile
His Phe Ser Gly His Val Phe Thr Val Arg 1250 1255 1260Lys Lys Glu
Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly 1265 1270 1275Val
Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp 1280 1285
1290Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser
1295 1300 1305Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro
Leu Gly 1310 1315 1320Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
Thr Ala Ser Gly 1325 1330 1335Gln Tyr Gly Gln Trp Ala Pro Lys Leu
Ala Arg Leu His Tyr Ser 1340 1345 1350Gly Ser Ile Asn Ala Trp Ser
Thr Lys Glu Pro Phe Ser Trp Ile 1355 1360 1365Lys Val Asp Leu Leu
Ala Pro Met Ile Ile His Gly Ile Lys Thr 1370 1375 1380Gln Gly Ala
Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe 1385 1390 1395Ile
Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg 1400 1405
1410Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp
1415 1420 1425Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile
Ile Ala 1430 1435 1440Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser
Ile Arg Ser Thr 1445 1450 1455Leu Arg Met Glu Leu Met Gly Cys Asp
Leu Asn Ser Cys Ser Met 1460 1465 1470Pro Leu Gly Met Glu Ser Lys
Ala Ile Ser Asp Ala Gln Ile Thr 1475 1480 1485Ala Ser Ser Tyr Phe
Thr Asn Met Phe Ala Thr Trp Ser Pro Ser 1490 1495 1500Lys Ala Arg
Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro 1505 1510 1515Gln
Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys 1520 1525
1530Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu
1535 1540 1545Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser
Ser Gln 1550 1555 1560Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn
Gly Lys Val Lys 1565 1570 1575Val Phe Gln Gly Asn Gln Asp Ser Phe
Thr Pro Val Val Asn Ser 1580 1585 1590Leu Asp Pro Pro Leu Leu Thr
Arg Tyr Leu Arg Ile His Pro Gln 1595 1600 1605Ser Trp Val His Gln
Ile Ala Leu Arg Met Glu Val Leu Gly Cys 1610 1615 1620Glu Ala Gln
Asp Leu Tyr 16253251DNAArtificial sequencemodified promoter
sequence 3tgtttgctgc ttgcaatgtt tgcccatttt agggtggaca caggacgctg
tggtttctga 60gccagggggc gactcagatc ccagccagtg gacttagccc ctgtttgctc
ctccgataac 120tggggtgacc ttggttaata ttcaccagca gcctcccccg
ttgcccctct ggatccactg 180cttaaatacg gacgaggaca gggccctgtc
tcctcagctt caggcaccac cactgacctg 240ggacagtgaa t
25145061DNAArtificial sequenceCodon optimised modified factor VIII
nucleotide sequence 4atgcagatcg agctgtccac ctgctttttt ctgtgcctgc
tgcggttctg cttcagcgcc 60acccggcggt actacctggg cgccgtggag ctgtcctggg
actacatgca gagcgacctg 120ggcgagctgc ccgtggacgc ccggttcccc
cccagagtgc ccaagagctt ccccttcaac 180accagcgtgg tgtacaagaa
aaccctgttc gtggagttca ccgaccacct gttcaatatc 240gccaagccca
ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac
300gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct
gcacgccgtg 360ggcgtgagct actggaaggc cagcgagggc gccgagtacg
acgaccagac cagccagcgg 420gagaaagaag atgacaaggt gttccctggc
ggcagccaca cctacgtgtg gcaggtgctg 480aaagaaaacg gccccatggc
ctccgacccc ctgtgcctga cctacagcta cctgagccac 540gtggacctgg
tgaaggacct gaacagcggc ctgatcggcg ctctgctcgt ctgccgggag
600ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct
gttcgccgtg 660ttcgacgagg gcaagagctg gcacagcgag acaaagaaca
gcctgatgca ggaccgggac 720gccgcctctg ccagagcctg gcccaagatg
cacaccgtga acggctacgt gaacagaagc 780ctgcccggcc tgattggctg
ccaccggaag agcgtgtact ggcacgtgat cggcatgggc 840accacacccg
aggtgcacag catctttctg gaagggcaca cctttctggt ccggaaccac
900cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac
actgctgatg 960gacctgggcc agttcctgct gttttgccac atcagctctc
accagcacga cggcatggaa 1020gcctacgtga aggtggactc ttgccccgag
gaaccccagc tgcggatgaa gaacaacgag 1080gaagccgagg actacgacga
cgacctgacc gacagcgaga tggacgtggt gcggttcgac 1140gacgacaaca
gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc
1200tgggtgcact atatcgccgc cgaggaagag gactgggact acgcccccct
ggtgctggcc 1260cccgacgaca gaagctacaa gagccagtac ctgaacaatg
gcccccagcg gatcggccgg 1320aagtacaaga aagtgcggtt catggcctac
accgacgaga cattcaagac ccgggaggcc 1380atccagcacg agagcggcat
cctgggcccc ctgctgtacg gcgaagtggg cgacacactg 1440ctgatcatct
tcaagaacca ggctagccgg ccctacaaca tctaccccca cggcatcacc
1500gacgtgcggc ccctgtacag caggcggctg cccaagggcg tgaagcacct
gaaggacttc 1560cccatcctgc ccggcgagat cttcaagtac aagtggaccg
tgaccgtgga ggacggcccc 1620accaagagcg accccagatg cctgacccgg
tactacagca gcttcgtgaa catggaacgg 1680gacctggcct ccgggctgat
cggacctctg ctgatctgct acaaagaaag cgtggaccag 1740cggggcaacc
agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag
1800aaccggtcct ggtatctgac cgagaacatc cagcggtttc tgcccaaccc
tgccggcgtg 1860cagctggaag atcccgagtt ccaggccagc aacatcatgc
actccatcaa tggctacgtg 1920ttcgactctc tgcagctctc cgtgtgtctg
cacgaggtgg cctactggta catcctgagc 1980atcggcgccc agaccgactt
cctgagcgtg ttcttcagcg gctacacctt caagcacaag 2040atggtgtacg
aggacaccct gaccctgttc cctttcagcg gcgagacagt gttcatgagc
2100atggaaaacc ccggcctgtg gattctgggc tgccacaaca gcgacttccg
gaaccggggc 2160atgaccgccc tgctgaaggt gtccagctgc gacaagaaca
ccggcgacta ctacgaggac 2220agctacgagg atatcagcgc ctacctgctg
tccaagaaca acgccatcga accccggagc 2280ttcagccaga acccccccgt
gctgacgcgt agcttcagcc agaacagccg gcaccccagc 2340acccggcaga
agcagttcaa cgccaccacc atccccgaga acgacatcga gaaaaccgac
2400ccttggtttg cccaccggac ccccatgccc aagatccaga acgtgtccag
cagcgacctg 2460ctgatgctgc tgcggcagag ccccacccct cacggcctga
gcctgagcga cctgcaggaa 2520gccaagtacg agacattcag cgacgacccc
agccctggcg ccatcgacag caacaacagc 2580ctgtccgaga tgacccactt
ccggccccag ctgcaccaca gcggcgacat ggtgttcacc 2640cccgagagcg
gcctgcagct gcggctgaac gagaagctgg gcaccaccgc cgccaccgag
2700ctgaagaagc tggacttcaa ggtctccagc accagcaaca acctgatcag
caccatcccc 2760agcgacaacc tggccgctgg caccgacaac accagcagcc
tgggccctcc cagcatgccc 2820gtgcactacg acagccagct ggacaccacc
ctgttcggca agaagtccag ccccctgacc 2880gagtccggcg gacccctgtc
cctgagcgag gaaaacaacg acagcaagct gctggaaagc 2940ggcctgatga
acagccagga aagcagctgg ggcaagaatg tgtccagcac gcgtcaccag
3000cgggagatca cccggacaac cctgcagtcc gaccaggaag agatcgatta
cgacgacacc 3060atcagcgtgg agatgaagaa agaggatttc gatatctacg
acgaggacga gaaccagagc 3120cccagaagct tccagaagaa aacccggcac
tacttcattg ccgccgtgga gaggctgtgg 3180gactacggca tgagttctag
cccccacgtg ctgcggaacc gggcccagag cggcagcgtg 3240ccccagttca
agaaagtggt gttccaggaa ttcacagacg gcagcttcac ccagcctctg
3300tatagaggcg agctgaacga gcacctgggg ctgctggggc cctacatcag
ggccgaagtg 3360gaggacaaca tcatggtgac cttccggaat caggccagca
gaccctactc cttctacagc 3420agcctgatca gctacgaaga ggaccagcgg
cagggcgccg aaccccggaa gaacttcgtg 3480aagcccaacg aaaccaagac
ctacttctgg aaagtgcagc accacatggc ccccaccaag 3540gacgagttcg
actgcaaggc ctgggcctac ttcagcgacg tggatctgga aaaggacgtg
3600cactctggac tgattggccc actcctggtc tgccacacta acaccctcaa
ccccgcccac 3660ggccgccagg tgaccgtgca ggaattcgcc ctgttcttca
ccatcttcga cgagacaaag 3720tcctggtact tcaccgagaa tatggaacgg
aactgcagag ccccctgcaa catccagatg 3780gaagatccta ccttcaaaga
gaactaccgg ttccacgcca tcaacggcta catcatggac 3840accctgcctg
gcctggtgat ggcccaggac cagagaatcc ggtggtatct gctgtccatg
3900ggcagcaacg agaatatcca cagcatccac ttcagcggcc acgtgttcac
cgtgcggaag 3960aaagaagagt acaagatggc cctgtacaac ctgtaccccg
gcgtgttcga gacagtggag 4020atgctgccca gcaaggccgg catctggcgg
gtggagtgtc tgatcggcga gcacctgcac 4080gctggcatga gcaccctgtt
tctggtgtac agcaacaagt gccagacccc actgggcatg 4140gcctctggcc
acatccggga cttccagatc accgcctccg gccagtacgg ccagtgggcc
4200cccaagctgg ccagactgca ctacagcggc agcatcaacg cctggtccac
caaagagccc 4260ttcagctgga tcaaggtgga cctgctggcc cctatgatca
tccacggcat taagacccag 4320ggcgccaggc agaagttcag cagcctgtac
atcagccagt tcatcatcat gtacagcctg 4380gacggcaaga agtggcagac
ctaccggggc aacagcaccg gcaccctgat ggtgttcttc 4440ggcaatgtgg
acagcagcgg catcaagcac aacatcttca acccccccat cattgcccgg
4500tacatccggc tgcaccccac ccactacagc attagatcca cactgagaat
ggaactgatg 4560ggctgcgacc tgaactcctg cagcatgcct ctgggcatgg
aaagcaaggc catcagcgac 4620gcccagatca cagccagcag ctacttcacc
aacatgttcg ccacctggtc cccctccaag 4680gccaggctgc acctgcaggg
ccggtccaac gcctggcggc ctcaggtcaa caaccccaaa 4740gaatggctgc
aggtggactt tcagaaaacc atgaaggtga ccggcgtgac cacccagggc
4800gtgaaaagcc tgctgaccag catgtacgtg aaagagtttc tgatcagcag
ctctcaggat 4860ggccaccagt ggaccctgtt ctttcagaac ggcaaggtga
aagtgttcca gggcaaccag 4920gactccttca cccccgtggt gaactccctg
gacccccccc tgctgacccg ctacctgaga 4980atccaccccc agtcttgggt
gcaccagatc gccctcagga tggaagtcct gggatgtgag 5040gcccaggatc
tgtactgatg a 506154377DNAArtificial sequenceCodon optimised
modified factor VIII nucleotide sequence 5atgcagatcg agctgtccac
ctgctttttt ctgtgcctgc tgcggttctg cttcagcgcc 60acccggcggt actacctggg
cgccgtggag ctgtcctggg actacatgca gagcgacctg 120ggcgagctgc
ccgtggacgc ccggttcccc cccagagtgc ccaagagctt ccccttcaac
180accagcgtgg tgtacaagaa aaccctgttc gtggagttca ccgaccacct
gttcaatatc 240gccaagccca ggcccccctg gatgggcctg ctgggcccca
ccatccaggc cgaggtgtac 300gacaccgtgg tgatcaccct gaagaacatg
gccagccacc ccgtgagcct gcacgccgtg 360ggcgtgagct actggaaggc
cagcgagggc gccgagtacg acgaccagac cagccagcgg 420gagaaagaag
atgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg
480aaagaaaacg gccccatggc ctccgacccc ctgtgcctga cctacagcta
cctgagccac 540gtggacctgg tgaaggacct gaacagcggc ctgatcggcg
ctctgctcgt ctgccgggag 600ggcagcctgg ccaaagagaa aacccagacc
ctgcacaagt tcatcctgct gttcgccgtg 660ttcgacgagg gcaagagctg
gcacagcgag acaaagaaca gcctgatgca ggaccgggac 720gccgcctctg
ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacagaagc
780ctgcccggcc tgattggctg ccaccggaag agcgtgtact ggcacgtgat
cggcatgggc 840accacacccg aggtgcacag catctttctg gaagggcaca
cctttctggt ccggaaccac 900cggcaggcca gcctggaaat cagccctatc
accttcctga ccgcccagac actgctgatg 960gacctgggcc agttcctgct
gttttgccac atcagctctc accagcacga cggcatggaa 1020gcctacgtga
aggtggactc ttgccccgag gaaccccagc tgcggatgaa gaacaacgag
1080gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt
gcggttcgac 1140gacgacaaca gccccagctt catccagatc agaagcgtgg
ccaagaagca ccccaagacc 1200tgggtgcact atatcgccgc cgaggaagag
gactgggact acgcccccct ggtgctggcc 1260cccgacgaca gaagctacaa
gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320aagtacaaga
aagtgcggtt catggcctac accgacgaga cattcaagac ccgggaggcc
1380atccagcacg agagcggcat cctgggcccc ctgctgtacg gcgaagtggg
cgacacactg 1440ctgatcatct tcaagaacca ggctagccgg ccctacaaca
tctaccccca cggcatcacc 1500gacgtgcggc ccctgtacag caggcggctg
cccaagggcg tgaagcacct gaaggacttc 1560cccatcctgc ccggcgagat
cttcaagtac aagtggaccg tgaccgtgga ggacggcccc 1620accaagagcg
accccagatg cctgacccgg tactacagca gcttcgtgaa catggaacgg
1680gacctggcct ccgggctgat cggacctctg ctgatctgct acaaagaaag
cgtggaccag 1740cggggcaacc agatcatgag cgacaagcgg aacgtgatcc
tgttcagcgt gttcgatgag 1800aaccggtcct ggtatctgac cgagaacatc
cagcggtttc tgcccaaccc tgccggcgtg 1860cagctggaag atcccgagtt
ccaggccagc aacatcatgc actccatcaa tggctacgtg 1920ttcgactctc
tgcagctctc cgtgtgtctg cacgaggtgg cctactggta catcctgagc
1980atcggcgccc agaccgactt cctgagcgtg ttcttcagcg gctacacctt
caagcacaag 2040atggtgtacg aggacaccct gaccctgttc cctttcagcg
gcgagacagt gttcatgagc 2100atggaaaacc ccggcctgtg gattctgggc
tgccacaaca gcgacttccg gaaccggggc 2160atgaccgccc tgctgaaggt
gtccagctgc gacaagaaca ccggcgacta ctacgaggac 2220agctacgagg
atatcagcgc ctacctgctg tccaagaaca acgccatcga accccggagc
2280ttcagccaga acccccccgt gctgacgcgt caccagcggg agatcacccg
gacaaccctg 2340cagtccgacc aggaagagat cgattacgac gacaccatca
gcgtggagat gaagaaagag 2400gatttcgata tctacgacga ggacgagaac
cagagcccca gaagcttcca gaagaaaacc 2460cggcactact tcattgccgc
cgtggagagg ctgtgggact acggcatgag ttctagcccc 2520cacgtgctgc
ggaaccgggc ccagagcggc agcgtgcccc agttcaagaa agtggtgttc
2580caggaattca cagacggcag cttcacccag cctctgtata gaggcgagct
gaacgagcac 2640ctggggctgc tggggcccta catcagggcc gaagtggagg
acaacatcat ggtgaccttc 2700cggaatcagg ccagcagacc ctactccttc
tacagcagcc tgatcagcta cgaagaggac 2760cagcggcagg gcgccgaacc
ccggaagaac ttcgtgaagc ccaacgaaac caagacctac 2820ttctggaaag
tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg
2880gcctacttca gcgacgtgga tctggaaaag gacgtgcact ctggactgat
tggcccactc 2940ctggtctgcc acactaacac cctcaacccc gcccacggcc
gccaggtgac cgtgcaggaa 3000ttcgccctgt tcttcaccat cttcgacgag
acaaagtcct ggtacttcac cgagaatatg 3060gaacggaact gcagagcccc
ctgcaacatc cagatggaag atcctacctt caaagagaac 3120taccggttcc
acgccatcaa cggctacatc atggacaccc tgcctggcct ggtgatggcc
3180caggaccaga gaatccggtg gtatctgctg tccatgggca gcaacgagaa
tatccacagc 3240atccacttca gcggccacgt gttcaccgtg cggaagaaag
aagagtacaa gatggccctg 3300tacaacctgt accccggcgt gttcgagaca
gtggagatgc tgcccagcaa ggccggcatc 3360tggcgggtgg agtgtctgat
cggcgagcac ctgcacgctg gcatgagcac cctgtttctg 3420gtgtacagca
acaagtgcca gaccccactg ggcatggcct ctggccacat ccgggacttc
3480cagatcaccg cctccggcca gtacggccag tgggccccca agctggccag
actgcactac 3540agcggcagca tcaacgcctg gtccaccaaa gagcccttca
gctggatcaa ggtggacctg 3600ctggccccta tgatcatcca cggcattaag
acccagggcg ccaggcagaa gttcagcagc 3660ctgtacatca gccagttcat
catcatgtac agcctggacg gcaagaagtg gcagacctac 3720cggggcaaca
gcaccggcac cctgatggtg ttcttcggca atgtggacag cagcggcatc
3780aagcacaaca tcttcaaccc ccccatcatt gcccggtaca tccggctgca
ccccacccac 3840tacagcatta gatccacact gagaatggaa ctgatgggct
gcgacctgaa ctcctgcagc 3900atgcctctgg gcatggaaag caaggccatc
agcgacgccc agatcacagc cagcagctac 3960ttcaccaaca tgttcgccac
ctggtccccc tccaaggcca ggctgcacct gcagggccgg 4020tccaacgcct
ggcggcctca ggtcaacaac cccaaagaat ggctgcaggt ggactttcag
4080aaaaccatga aggtgaccgg cgtgaccacc cagggcgtga aaagcctgct
gaccagcatg 4140tacgtgaaag agtttctgat cagcagctct caggatggcc
accagtggac cctgttcttt 4200cagaacggca aggtgaaagt gttccagggc
aaccaggact ccttcacccc cgtggtgaac 4260tccctggacc cccccctgct
gacccgctac ctgagaatcc acccccagtc ttgggtgcac 4320cagatcgccc
tcaggatgga agtcctggga tgtgaggccc aggatctgta ctgatga
437764980DNAArtificial sequenceCodon optimised modified factor VIII
nucleotide sequence 6atgcagatcg agctgtccac ctgctttttt ctgtgcctgc
tgcggttctg cttcagcgcc 60acccggcggt actacctggg cgccgtggag ctgtcctggg
actacatgca gagcgacctg 120ggcgagctgc ccgtggacgc ccggttcccc
cccagagtgc ccaagagctt ccccttcaac 180accagcgtgg tgtacaagaa
aaccctgttc gtggagttca ccgaccacct gttcaatatc 240gccaagccca
ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac
300gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct
gcacgccgtg 360ggcgtgagct actggaaggc cagcgagggc gccgagtacg
acgaccagac cagccagcgg 420gagaaagaag atgacaaggt gttccctggc
ggcagccaca cctacgtgtg gcaggtgctg 480aaagaaaacg gccccatggc
ctccgacccc ctgtgcctga cctacagcta cctgagccac 540gtggacctgg
tgaaggacct gaacagcggc ctgatcggcg ctctgctcgt ctgccgggag
600ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct
gttcgccgtg 660ttcgacgagg gcaagagctg gcacagcgag acaaagaaca
gcctgatgca ggaccgggac 720gccgcctctg ccagagcctg gcccaagatg
cacaccgtga acggctacgt gaacagaagc 780ctgcccggcc tgattggctg
ccaccggaag agcgtgtact ggcacgtgat cggcatgggc 840accacacccg
aggtgcacag catctttctg gaagggcaca cctttctggt ccggaaccac
900cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac
actgctgatg 960gacctgggcc agttcctgct gttttgccac atcagctctc
accagcacga cggcatggaa 1020gcctacgtga aggtggactc ttgccccgag
gaaccccagc tgcggatgaa gaacaacgag 1080gaagccgagg actacgacga
cgacctgacc gacagcgaga tggacgtggt gcggttcgac 1140gacgacaaca
gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc
1200tgggtgcact atatcgccgc cgaggaagag gactgggact acgcccccct
ggtgctggcc 1260cccgacgaca gaagctacaa gagccagtac ctgaacaatg
gcccccagcg gatcggccgg 1320aagtacaaga aagtgcggtt catggcctac
accgacgaga cattcaagac ccgggaggcc 1380atccagcacg agagcggcat
cctgggcccc ctgctgtacg gcgaagtggg cgacacactg 1440ctgatcatct
tcaagaacca ggctagccgg ccctacaaca tctaccccca cggcatcacc
1500gacgtgcggc ccctgtacag caggcggctg cccaagggcg tgaagcacct
gaaggacttc 1560cccatcctgc ccggcgagat cttcaagtac aagtggaccg
tgaccgtgga ggacggcccc 1620accaagagcg accccagatg cctgacccgg
tactacagca gcttcgtgaa catggaacgg 1680gacctggcct ccgggctgat
cggacctctg ctgatctgct acaaagaaag cgtggaccag 1740cggggcaacc
agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag
1800aaccggtcct ggtatctgac cgagaacatc cagcggtttc tgcccaaccc
tgccggcgtg 1860cagctggaag atcccgagtt ccaggccagc aacatcatgc
actccatcaa tggctacgtg 1920ttcgactctc tgcagctctc cgtgtgtctg
cacgaggtgg cctactggta catcctgagc 1980atcggcgccc agaccgactt
cctgagcgtg ttcttcagcg gctacacctt caagcacaag 2040atggtgtacg
aggacaccct gaccctgttc cctttcagcg gcgagacagt gttcatgagc
2100atggaaaacc ccggcctgtg gattctgggc tgccacaaca gcgacttccg
gaaccggggc 2160atgaccgccc tgctgaaggt gtccagctgc gacaagaaca
ccggcgacta ctacgaggac 2220agctacgagg atatcagcgc ctacctgctg
tccaagaaca acgccatcga accccggagc 2280ttcagccaga acccccccgt
gctgacgcgt ggcatgcggc ccagaaacca caccgtgctg 2340ctgaaagtgt
gcaacaaggt gcccatcaac aacagcaccc acagcgtgaa caccagcacc
2400ctgaacgcca ccgacggcag gcggtgctac ctgaagaagg tgaccctgac
cccccctggc 2460ggcgaggacg ccaacatcac ctctggcgag cgcatccccg
aggacatcct ggaagagctg 2520cagctggatg gcgaagtgac agccgcccct
tccggcatcc tgagagtggg cgagcaggcc 2580ctgcggcgga gaaggcaggc
cgaggacaac tccaccgact tcgacatcta cagcggcgac 2640gagggcggcg
aagagcggag cgagatccag gaacgcccag ccgagaaccg gaccaccgcc
2700aacgccaccg gcatcaaccg gaccaaagag ctggaagagg gcaacgagat
cctgctgacc 2760ggcaaccccg gcaacagcaa gcggagcttc accgtggagc
ccgtgaccag cgaggaaatg 2820gtgcagaact acatcatgtc tgaggaagag
aagctgaagc cccaccatca gcagcccggc 2880aacgtggccg tgaacgagct
gtctgtgacg cgtcaccagc gggagatcac ccggacaacc 2940ctgcagtccg
accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa
3000gaggatttcg atatctacga cgaggacgag aaccagagcc ccagaagctt
ccagaagaaa 3060acccggcact acttcattgc cgccgtggag aggctgtggg
actacggcat gagttctagc 3120ccccacgtgc tgcggaaccg ggcccagagc
ggcagcgtgc cccagttcaa gaaagtggtg 3180ttccaggaat tcacagacgg
cagcttcacc cagcctctgt atagaggcga gctgaacgag 3240cacctggggc
tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc
3300ttccggaatc aggccagcag accctactcc ttctacagca gcctgatcag
ctacgaagag 3360gaccagcggc agggcgccga accccggaag aacttcgtga
agcccaacga aaccaagacc 3420tacttctgga aagtgcagca ccacatggcc
cccaccaagg acgagttcga ctgcaaggcc 3480tgggcctact tcagcgacgt
ggatctggaa aaggacgtgc actctggact gattggccca 3540ctcctggtct
gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag
3600gaattcgccc tgttcttcac catcttcgac gagacaaagt cctggtactt
caccgagaat 3660atggaacgga actgcagagc cccctgcaac atccagatgg
aagatcctac cttcaaagag 3720aactaccggt tccacgccat caacggctac
atcatggaca ccctgcctgg cctggtgatg 3780gcccaggacc agagaatccg
gtggtatctg ctgtccatgg gcagcaacga gaatatccac 3840agcatccact
tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc
3900ctgtacaacc tgtaccccgg cgtgttcgag acagtggaga tgctgcccag
caaggccggc 3960atctggcggg tggagtgtct gatcggcgag cacctgcacg
ctggcatgag caccctgttt 4020ctggtgtaca gcaacaagtg ccagacccca
ctgggcatgg cctctggcca catccgggac 4080ttccagatca ccgcctccgg
ccagtacggc cagtgggccc ccaagctggc cagactgcac 4140tacagcggca
gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac
4200ctgctggccc ctatgatcat ccacggcatt aagacccagg gcgccaggca
gaagttcagc 4260agcctgtaca tcagccagtt catcatcatg tacagcctgg
acggcaagaa gtggcagacc 4320taccggggca acagcaccgg caccctgatg
gtgttcttcg gcaatgtgga cagcagcggc 4380atcaagcaca acatcttcaa
cccccccatc attgcccggt acatccggct gcaccccacc 4440cactacagca
ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc
4500agcatgcctc tgggcatgga aagcaaggcc atcagcgacg cccagatcac
agccagcagc 4560tacttcacca acatgttcgc cacctggtcc ccctccaagg
ccaggctgca cctgcagggc 4620cggtccaacg cctggcggcc tcaggtcaac
aaccccaaag aatggctgca ggtggacttt 4680cagaaaacca tgaaggtgac
cggcgtgacc acccagggcg tgaaaagcct gctgaccagc 4740atgtacgtga
aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc
4800tttcagaacg gcaaggtgaa agtgttccag ggcaaccagg actccttcac
ccccgtggtg 4860aactccctgg acccccccct gctgacccgc tacctgagaa
tccaccccca gtcttgggtg 4920caccagatcg ccctcaggat ggaagtcctg
ggatgtgagg cccaggatct gtactgatga 498075336DNAArtificial
sequenceCodon optimised modified factor VIII nucleotide sequence
7tgtttgctgc ttgcaatgtt tgcccatttt agggtggaca caggacgctg tggtttctga
60gccagggggc gactcagatc ccagccagtg gacttagccc ctgtttgctc ctccgataac
120tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct
ggatccactg 180cttaaatacg gacgaggaca gggccctgtc tcctcagctt
caggcaccac cactgacctg 240ggacagtgaa tcgcggccgc caccatgcag
attgagctga gcacctgctt cttcctgtgc 300ctgctgaggt tctgcttctc
tgccaccagg agatactacc tgggggctgt ggagctgagc 360tgggactaca
tgcagtctga cctgggggag ctgcctgtgg atgccaggtt cccccccaga
420gtgcccaaga gcttcccctt caacacctct gtggtgtaca agaagaccct
gtttgtggag 480ttcactgacc acctgttcaa cattgccaag cccaggcccc
cctggatggg cctgctgggc 540cccaccatcc aggctgaggt gtatgacact
gtggtgatca ccctgaagaa catggccagc 600caccctgtga gcctgcatgc
tgtgggggtg agctactgga aggcctctga gggggctgag 660tatgatgacc
agaccagcca gagggagaag gaggatgaca aggtgttccc tgggggcagc
720cacacctatg tgtggcaggt gctgaaggag aatggcccca tggcctctga
ccccctgtgc 780ctgacctaca gctacctgag ccatgtggac ctggtgaagg
acctgaactc tggcctgatt 840ggggccctgc tggtgtgcag ggagggcagc
ctggccaagg agaagaccca gaccctgcac 900aagttcatcc tgctgtttgc
tgtgtttgat gagggcaaga gctggcactc tgaaaccaag 960aacagcctga
tgcaggacag ggatgctgcc tctgccaggg cctggcccaa gatgcacact
1020gtgaatggct atgtgaacag gagcctgcct ggcctgattg gctgccacag
gaagtctgtg 1080tactggcatg tgattggcat gggcaccacc cctgaggtgc
acagcatctt cctggagggc 1140cacaccttcc tggtcaggaa ccacaggcag
gccagcctgg agatcagccc catcaccttc 1200ctgactgccc agaccctgct
gatggacctg ggccagttcc tgctgttctg ccacatcagc 1260agccaccagc
atgatggcat ggaggcctat gtgaaggtgg acagctgccc tgaggagccc
1320cagctgagga tgaagaacaa tgaggaggct gaggactatg atgatgacct
gactgactct 1380gagatggatg tggtgaggtt tgatgatgac aacagcccca
gcttcatcca gatcaggtct 1440gtggccaaga agcaccccaa gacctgggtg
cactacattg ctgctgagga ggaggactgg 1500gactatgccc ccctggtgct
ggcccctgat gacaggagct acaagagcca gtacctgaac 1560aatggccccc
agaggattgg caggaagtac aagaaggtca ggttcatggc ctacactgat
1620gaaaccttca agaccaggga ggccatccag catgagtctg gcatcctggg
ccccctgctg 1680tatggggagg tgggggacac cctgctgatc atcttcaaga
accaggccag caggccctac 1740aacatctacc cccatggcat cactgatgtg
aggcccctgt acagcaggag gctgcccaag 1800ggggtgaagc acctgaagga
cttccccatc ctgcctgggg agatcttcaa gtacaagtgg 1860actgtgactg
tggaggatgg ccccaccaag tctgacccca ggtgcctgac cagatactac
1920agcagctttg tgaacatgga gagggacctg gcctctggcc tgattggccc
cctgctgatc 1980tgctacaagg agtctgtgga ccagaggggc aaccagatca
tgtctgacaa gaggaatgtg 2040atcctgttct ctgtgtttga tgagaacagg
agctggtacc tgactgagaa catccagagg 2100ttcctgccca accctgctgg
ggtgcagctg gaggaccctg agttccaggc cagcaacatc 2160atgcacagca
tcaatggcta tgtgtttgac agcctgcagc tgtctgtgtg cctgcatgag
2220gtggcctact ggtacatcct gagcattggg gcccagactg acttcctgtc
tgtgttcttc 2280tctggctaca ccttcaagca caagatggtg tatgaggaca
ccctgaccct gttccccttc 2340tctggggaga ctgtgttcat gagcatggag
aaccctggcc tgtggattct gggctgccac 2400aactctgact tcaggaacag
gggcatgact gccctgctga aagtctccag ctgtgacaag 2460aacactgggg
actactatga ggacagctat gaggacatct ctgcctacct gctgagcaag
2520aacaatgcca ttgagcccag gagcttcagc cagaacagca ggcaccccag
caccaggcag 2580aagcagttca atgccaccac catccctgag aatgacatag
agaagacaga cccatggttt 2640gcccaccgga cccccatgcc caagatccag
aatgtgagca gctctgacct gctgatgctg 2700ctgaggcaga gccccacccc
ccatggcctg agcctgtctg acctgcagga ggccaagtat 2760gaaaccttct
ctgatgaccc cagccctggg gccattgaca gcaacaacag cctgtctgag
2820atgacccact tcaggcccca gctgcaccac tctggggaca tggtgttcac
ccctgagtct 2880ggcctgcagc tgaggctgaa tgagaagctg ggcaccactg
ctgccactga gctgaagaag 2940ctggacttca aagtctccag caccagcaac
aacctgatca gcaccatccc ctctgacaac 3000ctggctgctg gcactgacaa
caccagcagc ctgggccccc ccagcatgcc tgtgcactat 3060gacagccagc
tggacaccac cctgtttggc aagaagagca gccccctgac tgagtctggg
3120ggccccctga gcctgtctga ggagaacaat gacagcaagc tgctggagtc
tggcctgatg 3180aacagccagg agagcagctg gggcaagaat gtgagcagca
gggagatcac caggaccacc 3240ctgcagtctg accaggagga gattgactat
gatgacacca tctctgtgga gatgaagaag 3300gaggactttg acatctacga
cgaggacgag aaccagagcc ccaggagctt ccagaagaag 3360accaggcact
acttcattgc tgctgtggag aggctgtggg actatggcat gagcagcagc
3420ccccatgtgc tgaggaacag ggcccagtct ggctctgtgc cccagttcaa
gaaggtggtg 3480ttccaggagt tcactgatgg cagcttcacc cagcccctgt
acagagggga gctgaatgag 3540cacctgggcc tgctgggccc ctacatcagg
gctgaggtgg aggacaacat catggtgacc 3600ttcaggaacc aggccagcag
gccctacagc ttctacagca gcctgatcag ctatgaggag 3660gaccagaggc
agggggctga gcccaggaag aactttgtga agcccaatga aaccaagacc
3720tacttctgga aggtgcagca ccacatggcc cccaccaagg
atgagtttga ctgcaaggcc 3780tgggcctact tctctgatgt ggacctggag
aaggatgtgc actctggcct gattggcccc 3840ctgctggtgt gccacaccaa
caccctgaac cctgcccatg gcaggcaggt gactgtgcag 3900gagtttgccc
tgttcttcac catctttgat gaaaccaaga gctggtactt cactgagaac
3960atggagagga actgcagggc cccctgcaac atccagatgg aggaccccac
cttcaaggag 4020aactacaggt tccatgccat caatggctac atcatggaca
ccctgcctgg cctggtgatg 4080gcccaggacc agaggatcag gtggtacctg
ctgagcatgg gcagcaatga gaacatccac 4140agcatccact tctctggcca
tgtgttcact gtgaggaaga aggaggagta caagatggcc 4200ctgtacaacc
tgtaccctgg ggtgtttgag actgtggaga tgctgcccag caaggctggc
4260atctggaggg tggagtgcct gattggggag cacctgcatg ctggcatgag
caccctgttc 4320ctggtgtaca gcaacaagtg ccagaccccc ctgggcatgg
cctctggcca catcagggac 4380ttccagatca ctgcctctgg ccagtatggc
cagtgggccc ccaagctggc caggctgcac 4440tactctggca gcatcaatgc
ctggagcacc aaggagccct tcagctggat caaggtggac 4500ctgctggccc
ccatgatcat ccatggcatc aagacccagg gggccaggca gaagttcagc
4560agcctgtaca tcagccagtt catcatcatg tacagcctgg atggcaagaa
gtggcagacc 4620tacaggggca acagcactgg caccctgatg gtgttctttg
gcaatgtgga cagctctggc 4680atcaagcaca acatcttcaa cccccccatc
attgccagat acatcaggct gcaccccacc 4740cactacagca tcaggagcac
cctgaggatg gagctgatgg gctgtgacct gaacagctgc 4800agcatgcccc
tgggcatgga gagcaaggcc atctctgatg cccagatcac tgccagcagc
4860tacttcacca acatgtttgc cacctggagc cccagcaagg ccaggctgca
cctgcagggc 4920aggagcaatg cctggaggcc ccaggtcaac aaccccaagg
agtggctgca ggtggacttc 4980cagaagacca tgaaggtgac tggggtgacc
acccaggggg tgaagagcct gctgaccagc 5040atgtatgtga aggagttcct
gatcagcagc agccaggatg gccaccagtg gaccctgttc 5100ttccagaatg
gcaaggtgaa ggtgttccag ggcaaccagg acagcttcac ccctgtggtg
5160aacagcctgg acccccccct gctgaccaga tacctgagga ttcaccccca
gagctgggtg 5220caccagattg ccctgaggat ggaggtgctg ggctgtgagg
cccaggacct gtactgatcg 5280cgaataaaag atctttattt tcattagatc
tgtgtgttgg ttttttgtgt gatgca 5336842DNAArtificial sequenceSynthetic
construct 8agcttcagcc agaacccccc cgtgctgacg cgtcaccagc gg
42920DNAArtificial sequencePrimer 9tgtgtgcccg tctgttgtgt
201020DNAArtificial sequencePrimer 10gagtcctgcg tcgagagagc
201120DNAArtificial sequencePrimer 11cgcccgaaca gggacttgaa
201221DNAArtificial sequencePrimer 12aaaacgagca gtgacctgag g
211320DNAArtificial sequencePrimer 13ttcagtcatg ctgctagcgc
201422DNAArtificial sequencePrimer 14tgcacggaat ctcgtctcag tc
221525DNAArtificial sequencePrimer 15tcacccacaa gttgcccatc tacga
251625DNAArtificial sequencePrimer 16cagcggaacc gctcattgcc aatgg
251726DNAArtificial sequencePrimer 17atgccctccc ccatgccatc ctgcgt
261814PRTArtificial sequenceSynthetic construct 18Ser Phe Ser Gln
Asn Pro Pro Val Leu Thr Arg His Gln Arg1 5 101911PRTArtificial
sequenceSynthetic construct 19Ser Phe Ser Gln Asn Pro Pro Val Leu
Thr Arg1 5 10205PRTArtificial sequenceSynthetic construct 20Thr Arg
His Gln Arg1 5211670PRTArtificial sequenceModified Factor VIII
protein 21Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu
Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val
Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro
Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe
Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr
Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met
Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val
Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu
His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp
Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150
155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr
Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser
Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu
Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu
Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr
Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala
Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn
Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265
270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln
Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys
His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val
Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys
Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro
Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390
395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala
Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln
Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys
Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr
Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu
Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe
Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly
Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505
510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys
Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val
Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro
Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn
Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val
Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro
Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630
635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr
Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser
Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr
Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val
Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys
His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu
Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr
Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745
750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro
755 760 765Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu
Asn Asp 770 775 780Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr
Pro Met Pro Lys785 790 795 800Ile Gln Asn Val Ser Ser Ser Asp Leu
Leu Met Leu Leu Arg Gln Ser 805 810 815Pro Thr Pro His Gly Leu Ser
Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830Glu Thr Phe Ser Asp
Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840 845Ser Leu Ser
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly 850 855 860Asp
Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu865 870
875 880Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe
Lys 885 890 895Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro
Ser Asp Asn 900 905 910Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu
Gly Pro Pro Ser Met 915 920 925Pro Val His Tyr Asp Ser Gln Leu Asp
Thr Thr Leu Phe Gly Lys Lys 930 935 940Ser Ser Pro Leu Thr Glu Ser
Gly Gly Pro Leu Ser Leu Ser Glu Glu945 950 955 960Asn Asn Asp Ser
Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu 965 970 975Ser Ser
Trp Gly Lys Asn Val Ser Ser Arg Glu Ile Thr Arg Thr Thr 980 985
990Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val
995 1000 1005Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp
Glu Asn 1010 1015 1020Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg
His Tyr Phe Ile 1025 1030 1035Ala Ala Val Glu Arg Leu Trp Asp Tyr
Gly Met Ser Ser Ser Pro 1040 1045 1050His Val Leu Arg Asn Arg Ala
Gln Ser Gly Ser Val Pro Gln Phe 1055 1060 1065Lys Lys Val Val Phe
Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln 1070 1075 1080Pro Leu Tyr
Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 1085 1090 1095Pro
Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe 1100 1105
1110Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile
1115 1120 1125Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg
Lys Asn 1130 1135 1140Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe
Trp Lys Val Gln 1145 1150 1155His His Met Ala Pro Thr Lys Asp Glu
Phe Asp Cys Lys Ala Trp 1160 1165 1170Ala Tyr Phe Ser Asp Val Asp
Leu Glu Lys Asp Val His Ser Gly 1175 1180 1185Leu Ile Gly Pro Leu
Leu Val Cys His Thr Asn Thr Leu Asn Pro 1190 1195 1200Ala His Gly
Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe 1205 1210 1215Thr
Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met 1220 1225
1230Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro
1235 1240 1245Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly
Tyr Ile 1250 1255 1260Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln
Asp Gln Arg Ile 1265 1270 1275Arg Trp Tyr Leu Leu Ser Met Gly Ser
Asn Glu Asn Ile His Ser 1280 1285 1290Ile His Phe Ser Gly His Val
Phe Thr Val Arg Lys Lys Glu Glu 1295 1300 1305Tyr Lys Met Ala Leu
Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1310 1315 1320Val Glu Met
Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 1325 1330 1335Leu
Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu 1340 1345
1350Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly
1355 1360 1365His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr
Gly Gln 1370 1375 1380Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser
Gly Ser Ile Asn 1385 1390 1395Ala Trp Ser Thr Lys Glu Pro Phe Ser
Trp Ile Lys Val Asp Leu 1400 1405 1410Leu Ala Pro Met Ile Ile His
Gly Ile Lys Thr Gln Gly Ala Arg 1415 1420 1425Gln Lys Phe Ser Ser
Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr 1430 1435 1440Ser Leu Asp
Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr 1445 1450 1455Gly
Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile 1460 1465
1470Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg
1475 1480 1485Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg
Met Glu 1490 1495 1500Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met
Pro Leu Gly Met 1505 1510 1515Glu Ser Lys Ala Ile Ser Asp Ala Gln
Ile Thr Ala Ser Ser Tyr 1520 1525 1530Phe Thr Asn Met Phe Ala Thr
Trp Ser Pro Ser Lys Ala Arg Leu 1535 1540 1545His Leu Gln Gly Arg
Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1550 1555 1560Pro Lys Glu
Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val 1565 1570 1575Thr
Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met 1580 1585
1590Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln
1595 1600 1605Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe
Gln Gly 1610 1615 1620Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser
Leu Asp Pro Pro 1625 1630 1635Leu Leu Thr Arg Tyr Leu Arg Ile His
Pro Gln Ser Trp Val His 1640 1645 1650Gln Ile Ala Leu Arg Met Glu
Val Leu Gly Cys Glu Ala Gln Asp 1655 1660 1665Leu Tyr 1670
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References